TRANSFUSION

MEDICINE

Dr Zuhair Al-Barazanchi
2018
FORMS OF
IMMUNITY
- active - natural (post-infectious)
- artificial (post-vaccine)
- passive - natural (mother - foetus)
- artificial (antisera, gamma globulin)

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Non specific (natural, innate) Neutrophils Phagocytes
Specific (adaptive)
Eosinophils

Monocytes

Macrophages
Cells of the innate immunity
NK-cells Cytotoxic cells

Basophils Mediator cells 2

Mast cells
 ORGANS OF THE IMMUNE SYSTEM
 Primary lymphoid organs
 Bone marrow, thymus

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 Secondary lymphoid organs
 Lymph nodes, tonsils, spleen, Peyers patches, appendix

Recirculation

3
CHARACTERISTICS OF THE MECHANISMS OF NATURAL
IMMUNITY
 They are already present before the first encounter of an antigen
 They do not make any differences among antigens, the reaction to each
one is the same
 The re-exposition of an organism with the same antigen does not elicit
any quantitative or qualitative response compared to the first encounter

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4
 Characteristics of mechanisms of the
specific immunity
 Specificity
- The immune response is directed to one particular antigen
only, not to any other
 Diversity
- The immune system is able to distinguish the enormous
number of different antigens (109 – 1012)
 Self limitation: The immune response declines some time
after fulfilment its role

 Memory:
 Tolerance: The immune system distinguishes self molecules
from non self
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 M. Buc
6

oan et al. 2008

 THE FIRST LINE OF DEFENCE

 Skin (pH, fatty acids, epithelial cell desquamation)

 Mucosa of the upper respiratory tract (ciliated epithelium,

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cough, sneezing)

 Eye, oral cavity (lysozyme)

 Vagina (Lactobacilus Döderleini)

 Urine (acidic pH)

 Stomach (HCl)

 Normal intestinal flora (Escherichia coli) 7

MECHANISMS OF THE SECOND LINE OF
DEFENCE
 Cellular
components
- Phagocytes
- NK

 Humoral components
- Antibacterial proteins of the plasma
- Complement 8

- Carrier and inflammatory proteins

 MECHANISMS OF SPECIFIC
IMMUNITY
 Antibody mediated
Antibodies by themselves or by help of some other
components of IS destroy or neutralise antigens

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 Cell mediated
T cells are responsible for. They destroy targets foreign
cells by themselves or they „call“ other cells to help them
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 COMPLEMENT SYSTEM
> 30 proteins present in plasma and on cell surfaces that interact with each other to
produce biologically active inflammatory mediators that promote tissue injury and
eliminate blood and tissue pathogens.
Produced by hepatocytes (mainly), monocytes and epithelial cells of the
gastrointestinal an genitourinary tracts

Many components are proenzymes , which are functionally inactive until

proteolytic cleavage,which removes an inhibitory fragment and exposes the
active site.
Reaction starts with an enzyme cascade
-Complement proteins designated by “C” followed by numbers and
letters.
The proteins in this pathway are named C1-C9
• Components numbered in order of discovery not the order of the
reaction).

When complement is activated it is split into two parts

a – smaller of the two
B – larger part and usually the active part (except with factor 2)
Complement Activation
1. Classical Pathway – Begins with the formation of Ag-Ab complex
2. Alternative Pathway – Initiated by cell-surface constituents that are foreign to
the host
– Ab-independent
3. Lectin Pathway – Activated by the binding of mannose- binding lectin (MBL) to mannose
residues on glycoproteins or carbohydrates on the surface of microorganisms
– Ab-independent

13
Classical Pathway Begins with Ag-Ab Binding
soluble Ag-Ab* or bacteria-Ab* an anaphylatoxin or
↓ a mediator of
inflammation 
conformational changes in the anaphylaxis
Fc portion of Ig
↓
C4b C2a
expose a binding site on the
Fc portion for the C1q component of
the complement system

C1q molecule serves as recognition unit

For C1q to initiate the cascade it must attach to at least 2 Fc fragments, requires at
least 2 molecules of IgG or one molecule of IgM.

* complement-activating Abs:
IgM, IgG1, IgG2, IgG3 (human)
C1s cleaves or breakdown (hydrolyzes) C4 into C4a and C4b,
C1s hydrolyze C2 into C2b and C2a
↓ ___ 14
C4b and C2a form a C4b2a complex, also called C3
convertase,
referring to its role in converting the C3 into an active form.
 ____
C4b2a (C3 convertase) hydrolyzes C3 into C3b and C3a
↓ ____ ______
C3b binds to C4b2a and form C4b2a3b (C5 convertase)
_______ ↓
C4b2a3b cleaves C5 into C5b and C5a

opsonization inflammatory
responses
inflammatory
responses

15
CLASSICAL COMPLEMENT
PATHWAY

antibody
C1qrs

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C4b C6
C2a
C3b C7
C5b C8
C6
C9
C7
Bacteria, RBC C8
C9 C9
C9 C9
C9 C9
The Lectin Pathway Originates with Host Proteins Binding
Microbial Surfaces MBP

Lectin: proteins that bind to a carbohydrate

MBL (mannose-binding lectin): C4b C6
C2a
C3b C7
C5b C8
C6
Bacteria, RBC C9
C7
C8
C9 C9
C9 C9
C9 C9

- MBL is induced during inflammatory responses.

- MBL binds to the surface of a microbe
- resulting in the activation of MBL-associated
serine proteases (MASPs: MASP-1, MASP-2,
and MASP-3). Among these proteases, MASP-2
is a key enzyme that cleaves C4 and C2 . In
addition, MASP-1 was able to activate MASP-2
and MASP-3 as C1r activates C1s.
The Alternative Pathway
Ab-independent
- The activation of alternative pathway doesn’t
need Ab; thus, it is a component of the innate
immune system.
- It is initiated by cell-surface constituents that
are foreign to the host, e.g., bacterial cell wall.
- C1, C4 and C2 are not involved in the alternative
pathway.
- Four serum proteins, C3, factor B, factor D, and
properdin, are involved in this pathway.

Spontaneous conversion from C3 to C3b

occurs in body
Normally, C3b is very short lived and quickly
inactivated by proteins on the surface of the
body’s own cell walls
Bacteria or other foreign substances may 18
lack these surface proteins allowing C3b to
bind and stay active
 plasma C3, with an unstable thioester bond,
can be hydrolyzed spontaneously into C3a
and C3b.
C3b attaches to the surface of bacteria,
yeasts, viruses (or even host’s own cells ).
C3b + factor Bb with the aid of factor D and
++
Mg magnessium ion  C3bBb(C3 convertase)

Ba analogous to the C4b2a complex in the

stabilization( classical pathway
)of C3bBb
C3bBb3B will act as C5 convertase and the
process completes to MAC
C6
C3b C7
Bb
C5b C8
C6
C7 C9
C8
Bacteria, RBCs C9
C9 C9
C9 19 C9
C9 C9
SUMMARY OF COMPLEMENT
PATHWAYS
3 pathways for activation:

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1. classical: most specific (antibody
dependent activation, binds C1)
2. lectin binding: some specificity
(mannose binding protein, binds C4)
3. alternative: most primitive (non-
specific, auto-activation of C3)
 Biological Effects Mediated by Complement
2. Clearance of
1. Cell lysis immune complexes

The membrane-attack complex can

lyse a broad spectrum
of cells:
G(-) bacteria
parasites
viruses
erythrocytes
nucleated cells (tumor cells)
Because the activation of alternative
and lectin pathways is Ab-independent,
these pathways serve as important
innate immune defenses against
infectious microorganisms.

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3. Inflammatory response
- C3a, C4a, C5a (called anaphylatoxin) bind to complement receptors on mast
cells and basophils and induce degranulation with release of histamine and other
mediators.

- increased vascular permeability, extravasation, and chemo-attraction (induced

by C5a, C3a)

4. Opsonization
- C3b is the major opsonin of the
complement system,

5. Viral neutralization
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- The deposits of Ab and complement on viral particle neutralizes viral
infectivity by blocking attachment to susceptible host cells.
MHC
MHC = Major Histocombitibiliy Complex
MHC is a generic name
HLA = Human Leucocyte Antigen, eg SLA = Swine Leucocyte Antigen

They are linked cluster of genes, their products play a role in

intercellular recognition between self and nonself.

The MHC is a region of multiple loci that play major roles in

determining, whether transplanted tissue is accepted as self
(histocompatible) or rejected as foreign (histoincompatible)
26
Three types of molecules
encoded by the MHC:
Class I Class I MHC
Class II
All Nucleated cells
Class III

Class I MHC molecules are found

on all nucleated cells (not RBCs)

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Class II MHC molecules Class II MHC
found on: APC APCs,Dendriti
Dendritic cells c cells, B L,
Macrophages, MQ
B lymphocytes

Class III MHC molecules

Some complement components
Transporter proteins
RBCs
28
 CD4+T cell(Th) CD8+T cell(Tc)

T cell T cell
Receptor Receptor

Peptide Peptide
CD4 CD8

MHC MHC
Class II Class I

Antigen Presenting Cell Antigen Presenting Cell

 THE
THE INHERITANCE
INHERITANCE OF
OFMHC
MHC GENES
GENES

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1) MHC Class I and Class II molecules have many alleles (point mutations, gene
conversion, gene recombination, cross-over)

2) Collection of Class I + Class II alleles on one chromosome is called the

haplotype

3) MHC molecules are expressed co-dominantly: alleles from both chromosomes

are expressed in each cell.
CROSSING OVER
RESULTS IN NEW HAPLOTYPES

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 Polymorphism

Means there are multiple alleles at a gene locus of MHC in a

population so that an individual may have variable, multiple
allele CLASS I
POLYMORPHISM
MHC class I
No. of alleles locus
Major Antigens
2884 HLA A
3589 HLA B
2375 HLA C
Minor Antigens
15 HLA E
22 HLA F
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50 HLA G
Why so much polymorphism?: For the Survival of the species
CLASS II POLYMORPHISM

Number of alleles
(allotypes) Locus
26 HLA – DPA1
127 HLA – DPB1
34 HLA - DQA
90 HLA – DQB1
3 HLA - DRA
578 HLA - DRB1
83 HLA – DRB2-9
Relatively few alleles There are also HLA - DM and HLA – DO
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Ⅰ. ClassⅠgene region
 Classical HLA classⅠgenes-----------HLA-A,B,C
 Non-classical HLA classⅠgenes-----HLA-E,F,G
 MHC classⅠ chain related genes

Ⅱ. Class Ⅱ gene region

 Classical HLA classⅡ genes----------HLA-DP,DQ,DR
 Non-classical HLA classⅠgenes-----HLA-DM,DO

Ⅲ. Class Ⅲ gene region

 Complement genes-----------------------C4,C2,Bf
 Inflammation-associated genes------TNF,HSP70
Peptide Generation/ Binding to MHC Molecules: Class I vs. II

36
Class I Class II
• Generated by proteasome (cytosol) • Generated in endosomes/lysosome
• Restricted Peptide length, • 12-28 amino acids
9-10 amino acids
FUNCTION OF MHC
1. Key role in recognition of antigens
 Antigen specific T-cells do not recognize antigen in free or
soluble form or as intact protein. Instead, T-cells recognize
portions of protein antigens that have been fragmented into

37
peptides bond to HLA molecules.
 TCR recognizes antigenic peptides in association with MHC
molecules

 Tc cells (CD8) recognize peptides bound to class I MHC

molecules results in destruction of infected cell.
 Th cells (CD4)recognize peptides bound to class II MHC causes
activation (e.g., of antibody production by B-cells
2. Key role in cell-cell interactions in immune system
Clinical application of MHC
1- Organ transplantation the success of an organ transplant
depends on how closely the antigen patterns match, and they are
most likely to be similar when the donated organ or tissue comes
from a close blood relative of the recipient.

2- Paternity testing (forensic medicine). Since each parent can

only transmit one antigen to an offspring within a locus, the
number of different HLA antigens under A,B,C,D loci is very high,
the chance for two persons to have a common haplotype is
usually rare and less than 1 in 106 to 1010 population.

3- HLA-Ags are used to assessed anthropologist for detections

the origins of races and racial admixture.

4-Tissue typing is used to provide more evidence for the

diagnosis of the disease. For example, finding the B27 allele can
added a clearer diagnostic perspective for an individual with
ankylosing spondylitis.
 Association between MHC and diseases:
Over 40 diseases have well-established HLA association.
HLA and autoimmune diseases
Relative risk Diseases with increased risk HLA allele
12 Ankylosing spondylitis
14 Postgonococcal arthritis HLA-B27
15 Acute anterior uveitis
15 21-hydroxylase deficiency HLA-B47

2-3 Systemic lupus erythematosus HLA-DR2

14 Autoimmune hepatitis
10 Primary Sjögren syndrome
HLA-DR3
5 Diabetes mellitus type 1
2-3 Systemic lupus erythematosus
4 Rheumatoid arthritis
HLA-DR4
6 Diabetes mellitus type 1
HLA-DR3 and
15 Diabetes mellitus type 1
-DR4 combined
7 Coeliac disease HLA-DQ2 and HLA-DQ8
 ANTIGE
N
a substance that can stimulate an immune response (immunogenicity).

 Antigens:
- microbes
- Chemical substances

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- Proteins, saccharides
- transplanted organs and tissues
- modified self molecules and cells

Foreign

Distinct biochemical structure

Large molecular mass

Degradable 40
BIOCHEMICAL
STRUCTURE
Proteins, polypeptides

Polysaccharides
The best antigens
Glycoproteins, peptidoglycans

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Glycolipids

Lipids and native nucleic acids are not

antigenic

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 ANTIGEN DOSAGE
Dosage and the way of introduction into the body can affect
antigenicity

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 Low doses of an antigen do not induce an immune
response as they do not stimulate sufficient numbers of
cells of the immune system – low zone tolerance

 High doses of antigen do not induce an immune

response as cells of the immune system do not cope
with its processing: immunologic paralysis or high
zone tolerance
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Epitope(Antigenic
determinant)
is a part of the antigen to
which an antibody or a
lymphocyte is directed
Paratope: part of Ab which

Doan et al. 2008

binds the epitope

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The red cell membrane and chemistry of blood group
antigens)
The red cell membrane is composed of:
40% (w/w) lipids
10% carbohydrates,
50 % protein.
Blood group antigens can be:
1. integral proteins or glycoproteins
2. membrane glycolipids.
Blood group systems
The International Society of Blood Transfusion recognizes:
347 red cell surface antigens,
308 of which belong to one of 36 blood group systems
Each system represents either a single gene or two or three very closely linked homologous
genes.
Each system is genetically discrete from all others.
In addition, there are 39 antigens that have not been included in systems, owing to
inadequate genetic evidence.
Most blood groups are inherited as Mendelian characters, although environmental factors
may occasionally affect blood group expression.
The 36 systems represent a total of 41 genes:
MNS contains 3 loci,
Rh, Xg and Ch/Rg 2 loci each,
The other 32 systems each contain a single gene.

All of the genes are autosomal, except for:

XG and XK : on the X chromosome, and
CD99: on both the X and Y chromosomes.
All 41 genes have been identified and sequenced.
The functions of blood group antigens
Antibodies(Gamma globulins, Immunoglobulins)
Proteins produced by the B lymphocytes of the adaptive immune system in response to an
antigen for which they exhibit specific binding.

Depending on the origin of the antigenic stimulus,

antibodies can be:
(i) alloantibodies, when produced by an individual
against epitopes present in another individual of the
same species;
(ii) autoantibodies, when reactive with
determinants present on the individual’s own
antigens; and
(iii) Heteroantibodies (or xenoantibodies), when
produced against antigenic determinants present on
the cells of another species.
The first two are the antibodies encountered
routinely in the blood bank;
heteroantibodies can be used as antiglobulin sera or
typing reagents when raised in animals against
human antigens and are often present in reagents as
monoclonal antibodies.
Five classes of immunoglobulin
(IgG), IgM, IgA, IgD, IgE.
All these classes have the basic four‐chain structure but they differ in their heavy
chains, (γ, μ, α, δ, and ε, respectively)
Structural differences
1.Most pronounced in the Fc regions of the antibody classes and this leads to the
triggering of different effector functions on binding to antigen.
eg: IgM recognition of antigen might lead to complement activation,
IgE recognition (possibly of the same antigen) might lead to mast cell
degranulation and anaphylaxis (increased vascular permeability and
smooth muscle contraction).
2. Lead to differences in the polymerization state of the monomer unit. Thus, IgG and
IgE are generally monomeric, whereas IgM occurs as a pentamer. IgA occurs
predominantly as a monomer in serum and as a dimer in seromucous secretions.
Blood group antibodies
1. Naturally occurring vs. immune antibodies
Naturally occurring: produced without any obvious immunizing stimulus, such as
pregnancy or transfusion.
not present at birth and, in the case of anti-A,B, anti-A and anti-B, start to appear in
the serum at about 3–6 months of age.
ABO antibodies are probably produced in response to antigens of bacteria, viruses
and other substances that are inhaled or ingested.
immune antibodies: only produced after pregnancy or following transfusion or
injection of blood or blood group substances.
2. Cold vs. warm antibodies
Cold antibodies give higher agglutination titres at low temperatures (0–4ºC), and
many of them will not agglutinate red cells at 37ºC. Most naturally occurring
antibodies are cold reacting.
Some, such as naturally occurring anti-A,B, have a wide thermal range and will
react at 37 ◦C, activating complement and leading to red cell lysis. Yet, the titre will
be much higher at 0–4 ◦C.
Cold antibodies that fail to react above 30º C are of no clinical significance and can
be ignored for blood transfusion purposes.
Warm antibodis, mostly Immune, have a thermal optimum of 37º C. Any red cell
antibody reacting above 30º C should be considered potentially capable of
destroying red cells in vivo.
IgM vs IgG

IgM antibodies agglutinate red cells suspended in saline. They are often called
saline or directly agglutinating antibodies.

IgG antibodies do not usually agglutinate saline-suspended red cells. However,

lack of agglutination does not mean that the antibodies have not bound to their
antigen, and it can be shown that they have reacted by using antiglobulin reagents,
which facilitate agglutination of antibody-coated cells.

Most naturally occurring antibodies are IgM and preferentially cold-reacting,

whereas most immune antibodies are IgG and warm-reacting, although some may
be IgM. Exceptionally, very potent IgG antibodies may be directly agglutinating.
The ABO system
Discovered by the Austrian scientist Karl Landsteiner, in 1900
the first system to be recognized
the most important in transfusion and transplantation.
Almost every body over the age of about 6 months has clinically significant anti-A
and/or anti-B in their serum if they lack the corresponding antigens on their red cells.
GENES CONTROLLING ABO SYSTEM:

H genes
two genes, FUT1 and FUT2, on chromosome 19, are responsible for production of H antigen.
Both encode α1,2- fucosyl transferases that catalyse the transfer of fucose to the terminal
galactose of the H precursor chain.
FUT1 is active in mesodermally derived tissues, including haemopoietic tissues, and is
responsible for H expression on red cells.
Homozygosity for inactivating mutations in FUT1 gives rise to Bombay and related phenotypes.
FUT2 is responsible for the expression of H antigen in endodermally derived tissues, including
secretions, and hence is the gene responsible for ABH secretion.
Secretors are homozygous or heterozygous for an active FUT2;
ABO genes
ABO is located on the long arm of chromosome 9, comprises seven exons and
encodes the A and B glycosyl transferases.
Products of the A and B alleles differ by four amino acids encoded by exon 7, two of
which determine whether the enzyme has GalNAc-transferase (A) or Gal-
transferase (B) activity.
The majority of O alleles (called O1) resemble A, but have a single-base deletion in
exon 6, which creates a shift in the reading frame, truncating any putative
polypeptide.
About 3% of O alleles (called O2) have a single-nucleotide polymorphism (SNP) that
changes one of the vital amino acids in the catalytic site, inactivating the enzyme.
The A2 allele has a single-base deletion immediately before the usual termination
codon, creating a reading frameshift and abolitionof this stop codon. This produces
anA-transferasewith 21 extraneous amino acids on its C-terminus, reducing its
efficiency as a GalNAc-transferase.
ABH antigens

 On red cell membranes

 Predominantly glycoproteins,
 The majority: on the N-glycans of the
anion exchanger (band 3).
 Soluble ABH antigens are
glycoproteins.

Differences in the terminal sugars of

the glycoproteins and glycolipids
determine the specificity of these
antigens:
L-fucose (Fuc) for H,
L-fucose plus N-acetyl-D-
galactosamine (GalNAc) for A
L-fucose plus D-galactose (Gal) for B
Distribution of the A, B and H antigens
Often referred to as histo-blood group antigens because they are widely distributed
in the body.
Very important in transplantation.
Present on: white cells,
platelets
epidermal
other tissue cells.
Also present in the plasma, regardless of ABH secretor status, and in the saliva and
other secretions of ABH secretors
ABH secretor status
About 80% of Caucasian populations, ABH secretors, have H antigen, plus A or B
according to their ABO genotype, in a water soluble form in their body secretions.
The remaining 20% are non-secretors.
The ABH antigens
 Develop early in fetal life but do not increase much in strength during the
gestational period.

 The RBCs of the newborn have been estimated to carry anywhere from 25% to
50% of the number of antigenic sites found on the adult RBC. As a result, reactions
of newborn RBCs with ABO reagent antisera are frequently weaker than reactions
with adult cells.

 The expression of A and B antigens on the RBCs is fully developed by 2 to 4

years of age and remains constant throughout life.

 In addition to age, the phenotypic expression of ABH antigens may vary with race,
genetic interaction, and disease states.

H antigen
Group O cells have no antigens of the ABO system but do possess H antigen, the
precursor upon which the products of the ABO genes act.
The H gene (called FUT1) is on chromosome 19, whereas ABO is on chromosome
9.
The H antigen is present to some extent on almost all red cells, regardless of the
ABO group, but the amount of H antigen varies with the ABO group as follows:
O > A2 > A2B > B > A1 > A1B.
The A subgroups are generally more
common than B subgroups.
ABO subgroups
The weaker serologic reactivity of ABO
subgroups is attributed to the decreased
number of A and B antigen sites on their
red cells.
Classification into A1 and A2 phenotypes
accounts for 99% of all group A individuals.
The cells of approximately 80% of all group
A (or AB) individuals are A1
(or A1B),
the remaining 20% are A2 (or A2B) or
weaker subgroups.
The differences between A1 and A2 are
both quantitative and qualitative
The Bombay Phenotypes (Oh)
was first reported by Bhende in 1952 in Bombay, India.
 It represents the inheritance of a double dose of the h gene,(homozygous for
inactive FUT1 alleles (h/h)).
the ABO genes cannot be expressed, and ABH antigens cannot be formed, since
there is no H antigen made.
Their red cells are not agglutinated by anti-A or anti-B, regardless of ABO
genotype, but are not group O as they are also not agglutinated by anti-H.
The serum of Bombay subjects contains potent anti-H, anti-A and anti-B that will
only allow transfusion with red cells of the Bombay phenotype.
 Weakening of ABO antigens
1.Physiologically with aging

2. Weakening of the A antigen can occur in acute myeloid leukaemia. The

A antigen may revert to almost normal in remission.

3. Similar weakening of B, H and I has been described.

4. B-like antigens may be acquired by group A individuals who are suffering

from bowel infections, usually associated with carcinoma or strictures of the
large bowel. Red cells with an acquired B antigen are agglutinated by some
anti-B, including some monoclonal anti-B, but not by the patient’s own anti-
B.
Bacterial deacetylases convert N-acetylgalactosamine, the
immunodominant sugar of the A antigen, into galactosamine, a structure
similar to galactose, the immunodominant sugar of the B antigen.
Phenotype/Genotype
relationship
Antibodies of the ABO system
Anti-A, anti-B and anti-A,B

‘Naturally occurring’: arise in response to A- and B-like antigens present on

bacterial, viral or animal molecules.

Titers vary considerably with age, donot appear before 6 mo of age, reaching a
peak in young adults and then declining in old age.

Anti-A and anti-B have a wide thermal range. Although they are active at 37 ◦C,
they react better at lower temperatures.

almost entirely IgM(esp in gps A & B).

Antibodies from group O individuals, even before immunization, usually have

some IgG anti-A,B, an antibody that cross-reacts with both A and B structures.
Immune (IgG) Abs: mainly in group O pregnant ladies ABO HDFN are almost
always group O.
Immune anti-A,B are mainly IgG2, which does not cause HDFN because there are
no Fc receptors for IgG2 on the cells of the mononuclear phagocyte system.
 When the maternal serum contains potent IgG1 and/or IgG3 ABO antibodies,
HDFN may occur, although this is comparatively milder than Rh HDFN
Anti-A1
Anti-A1, reactive at roomtemperature (18–22 ◦C), can be found in the serum of:
1–8% of group A2 and
22–35% of group A2B persons.
Most of these antibodies are not of clinical importance because they do not
agglutinate A1 red cells at 30 ◦C and above.
Very rarely, anti-A1 able to react at 37 ◦C may lead to significant destruction of A1
red cells in vivo. The appropriate group A2 or A2B red cells should be provided in
these rare instances.

Anti-H
Several forms of anti-H exist:
1 Clinically significant ‘true’ anti-H occurs in the serum of the very rare
person with Bombay phenotype. It is active at 37 ◦C and only Bombay phenotype
blood may be transfused.
2 Anti-H and anti-HI, commonly found in the serum of group A1, B and A1B
persons, react much more strongly with adult than with cord red cells. Anti-H is
inhibited by secretor saliva; anti-HI is not. These antibodies do not usually
agglutinate O cells above 30 ◦C. Very occasionally, anti-H/anti-HI may cause
rapid destruction of at least some of the transfused O red cells in vivo. However,
these antibodies will not interfere with red cell survival if ABO identical units, i.e.
A1, B or A1B units, are transfused to A1, B orA1B recipients, respectively.
Antigen-Antibody Reactions
 ANTIGEN-ANTIBODY
RATIO

 The optimum ratio is 80 parts antibody to 1 part antigen.  There

are specific terms for variations in this ratio.
 Prozone - antibody excess: Antibodies saturating all antigen sites;
no antibodies forming cross-linkages between cells; no
agglutination
 Zone of equivalence: antibodies and antigens present in optimum
ratio, agglutination formed
 Zone of antigen excess (Post-zone): too many antigens - any
agglutination is hidden by masses of unagglutinated antigens
Detection of red cell antigen–antibody reactions
I. Agglutination(the clumping of particles: RBCs. The word agglutination comes
from the Latin agglutinare (glueing to).
The process that occurs if an antigen is mixed with its corresponding antibody
called isoagglutinin.
A. Direct agglutination: IgM antibodies will directly agglutinate saline-suspended
red cells of the appropriate phenotype.
used routinely for ABO grouping with polyclonal or monoclonal antibodies and RhD
typing with monoclonal antibodies.
B. Indirect agglutination
Apart from ABO, antibodies against most blood group antigens are IgG and
generally will not produce direct agglutination of red cells. Such antibodies can be
detected by the antiglobulin technique or with the aid of agents that enhance
agglutination, :

Enhancers/Potentiators
proteases, such as papain,
albumin and other colloids,
aggregating agents such as polybrene
and polyethylene glycol,
low ionic strength solutions (LISS)
Serum/cell mixture should incubate at Albumin
least 20 - 30 minutes;
doesn’t enhance warm autoantibodies
Incubation time of 10 minutes; LISS
lowers ionic strength allowing better
reaction; sensitive and quick!
Enhances warm autoantibodies; PEG
does not react well with insignificant Polyethylene
antibodies (IgM) glycol

Potentiators
GRADING
REACTIONS
II. Inhibition of agglutination
Expected agglutination reactions with known antigens and antibodies can be
neutralized by soluble antigens of the appropriate specificity.
Eg: the saliva of group A secretors inhibits the agglutination of group A cells by
anti-A.
Hydatid cyst fluid with P1PK activity can be used to confirm the presence of
anti-P1 in sera.
Soluble antigens produced by recombinant DNA technology are useful in
reference laboratories as aids to sorting out complex serological problems.
III. Haemolysis
After the incubation of red cells with fresh serum, red cell lysis indicates a positive
antigen–antibody reaction mediated by IgM complement-fixing antibodies.
A pink- or red-coloured supernatant after settling or centrifugation of red-cell–
antibody mixtures is an indication of hemolysis.

IV. Adsorption and elution tests

Specific antibodies can be removed from serum by adsorption on red cells
carrying the corresponding antigen.
Bound antibodies can be subsequently recovered from the washed, sensitized red
cells by elution with heat treatment, freeze-thawing, low pH or chloroquine
treatment.
 ABO
GROUPING

Forward Reverse
(Cell) (Serum)
grouping grouping
ABO GROUPING DISCREPANCIES
 Causes
 Newborns (the production of ABO
antibodies is not detectable until 4 to 6
months of age)
Elderly patients (the production of ABO
Group I Discrepancies antibodies is depressed)
 Leukemia (e.g.CLL) or lymphoma with
associated with unexpected reactions hypogammaglobulinemia
in the reverse grouping due to weakly  Immunosuppressive drugs that yield
reacting or missing antibodies. hypogammaglobulinemia
more common than those in the other  Congenital or acquired
groups listed. agammaglobulinemia or immunodeficiency
When a reaction in the serum grouping diseases
is weak or missing, a group I  Bone marrow or stem cell
discrepancy should be suspected, transplantations (patients develop
because, normally, RBC and serum hypogammaglobulinemia from therapy and
grouping reactions are very strong (4+). start producing a different RBC population
from that of the transplanted bone
marrow)
 Patients whose existing ABO antibodies
may have been diluted by plasma
transfusion or exchange transfusion.
 ABO subgroups
Group II Discrepancies
associated with unexpected reactions in the forward grouping due to weakly
reacting or missing antigens.
Probably the least frequently encountered.
Causes:
• Subgroups of A (or B) may be present.
• Leukemias may yield weakened A or B antigens and Hodgkin’s disease has been
reported in some cases to mimic the depression of antigens found in leukemia.

• The “acquired B” phenomenon will show weak reactions with anti-B antisera and
is most often associated with diseases of the digestive tract (e.g., cancer of the
colon). shows the ABO testing results of an acquired B phenomenon.
Group III Discrepancies
Caused by protein or plasma abnormalities and result in rouleaux formation or
pseudoagglutination, attributable to:
• Elevated levels of globulin from certain disease states, such as multiple myeloma,
Waldenström’s macroglobulinemia, other plasma cell dyscrasias, and certain
moderately advanced cases of Hodgkin’s lymphomas
• Elevated levels of fibrinogen
• Plasma expanders, such as dextran and polyvinylpyrrolidone
• Wharton’s jelly in cord blood samples
Knowledge of the following characteristics of
rouleaux helps in differentiation between
rouleaux and agglutination:
1. Cells have a "stacked coin" appearance
when viewed microscopically.
2. Rouleaux is observed in all tests
containing the patient's serum, including
the autologous control and the reverse
ABO typing.
3. Rouleaux does not interfere with the AHG
phase of testing because the patient's
serum is washed away prior to the addition
of the AHG reagent.
4. Unlike agglutination, rouleaux is dispersed
by the addition of 1 to 3 drops of saline to
the test tube.
5. Rouleaux is observed in all RBCs while
agglutination involves some of them with
others are not.
Group IV Discrepancies
due to miscellaneous problems and have the following causes:
• Cold reactive autoantibodies in which RBCs are so heavily coated with antibody
that they spontaneously agglutinate, independent of the specificity of the reagent
antibody

• Patient has circulating RBCs of more than one ABO group due to RBC transfusion
or marrow/stem cell transplant
• Unexpected ABO isoagglutinins
• Unexpected non-ABO alloantibodies
The Rh system

Was first discovered in 1937 by Karl Landsteiner and Alexander S.

Wiener.
The second most important blood group system in blood transfusion.
The 4th in discovery
A complex system
contains a total of 54 antigens, but D is the most important, owing to its high
immunogenicity and ability to cause severe HDFN and HTRs.
The next most important antigens are : c, C, E, e.
No d antigen: “d”= the absence of “D”
Proteins
Integral
Autosomal

D is considerably more immunogenic than the other Rh antigens, which have the
following order of immunogenicity:
c > E > e > C.

Depending on the DCEce nomenclature of Fisher: 8 haplotypes result

The original discovery of Rh antigens
Rhesus
monkey Rabbit/
RBCs Guinea pig

Anti
Agglutination Rhesus Ab

Rh + Human RBCs
Positive
85 %
No Agglutination

Rh
Negative
15 %
Molecular genetics of Rh
Rh genes and proteins
Rh antigens are encoded by two closely linked genes on chromosome 1 with 92%
sequence homology.
1.RHD encodes the D antigen
2.RHCE encodes the Cc and Ee antigens.

In Caucasian D-negative individuals,

the RHD gene is deleted,

in black and other populations,:

1.single- point mutation
2. partial deletions
3.
recombinations

In individuals with a weak D antigen

(Du), there is a quantitative
reduction in D antigen sites,
believed to arise from an
uncharacterised transcriptional
Rh antigens
The Rh antigens are named by:
1. The component antigens (e.g. DCe, dce)(Fisher’s)
2. Shorthand symbol (e.g. R = DCe, r = dce).
3. Wiener’s Rh-Hr terminology
4. Rosenfield’s numeric notation,
CDE nomenclature, derived from Fisher’s original theory, is recommended by a
World Health Organisation Expert Committee in the interest of simplicity and
uniformity.
Great racial and ethnic variation
in the frequency of Rh antigens.
How to convert Fisher symbols to shorthand symbols

D R
Rh haplotype notation
With (R/D)
Wiener Fisher-Race
C 1
R0 Dce
E 2
R1 DCe
ce º
R2 DcE
CE z
With (r/d) RZ DCE
C r’ r dce
E r” r′ dCe
CD ry r″ dcE
cd r ry dCE
Antibodies of the Rh system

Naturally occurring antibodies

Anti-E is often naturally occurring; about half may occur without a history of
pregnancy or transfusion.
Rarely, naturally occurring anti-D and anti-C are found.

Immune antibodies
Almost all.
The clinical importance of the Rh system lies in the readiness with which anti D
arises in D-negative subjects after stimulation with D-positive red cells by
pregnancy or transfusion.
About 20–30% of anti-D sera also appear to contain anti-C. Usually, this anti-C
is not a separable antibody and is probably more correctly called anti-G.
About 1–2% of anti-D sera also contain anti-E.
Anti-C (and anti-G) in the absence of anti-D is very uncommon.
The incidence of other Rh antibodies is much lower.
The Kell (Kell–Cellano )blood group system

was named after the first patient described with antibodies to K1, a pregnant
woman named Mrs. Kellacher in 1945. Mrs. Cellano was likewise a pregnant
woman with the first described antibodies to K2

consists of 35 antigens, including one triplet and four pairs of allelic antigens
K and k;
Kpa, Kpb and Kpc;
Jsa and Jsb;
K11 and K17;
K14 and K24
all of which represent amino acid substitutions in the Kell glycoprotein.

Three very closely linked sets of alleles are clinically important:

K (KEL1) and k (KEL2);
Kpa (KEL3), Kpb (KEL4) and Kpc (KEL21);
Jsa (KEL6) and Jsb (KEL7).

These antigens are encoded by alleles at the KEL locus on chromosome 7, but
their production also depends on genes at the KX locus on the X chromosome.
In European whites, the incidence of the K/k phenotypes, the most important
clinically, is as follows:

K+k− 0.2 %
K+k+ 8.7 %
K−k+ 91.1 %

K is rare in populations other than whites.

Jsa is present in about 20% of black people, but is extremely rare in other ethnic
groups.

Ko, a very rare null phenotype of the Kell system in which no Kell antigens are
expressed, results from homozygosity for a variety of inactivating mutations in
the KEL gene.

The Kell antigens are located on a glycoprotein that crosses the cell membrane
once.

The Kell protein has also recently been designated CD238

Anti-K
An important antibody in white populations;
Nearly always immune, IgG and complement-binding.
Causes severe HTRs and HDFN.
K stimulates the formation of anti-K in about 10% of K-negative people given one
unit of K-positive blood.
About 0.1% of all cases of HDFN are caused by anti- K.
HDFN caused by anti-K differs from Rh HDFN in that anti-K causes fetal anaemia
by suppression of erythropoiesis, rather than immune destruction of mature fetal
erythrocytes.
Explained by: Kell antigen is expressed by erythroid cells at a very early stage of
erythropoiesis :
anti-K would  destruction of early erythroid progenitors, before they become
haemoglobinized.
Anti-k
Very rare,
Reacting with 99.8% of random blood samples.
Always immune and has been incriminated in HDFN.

Most other Kell system antibodies are rare, best detected by the IAT and are
usually clinically significant.
 KEL XK
On Ch 7 on Ch X

Kell GP -------S—S------ Kx protein

The Kell glycoprotein is linked by a single disulfide bond to the Kx protein,

produced by an X-linked gene, XK.
Absence of Kx, resulting from hemizygosity in males of a deletion of XK or of XK
containing inactivating mutations, gives rise to
the McLeod syndrome, in which there is

no expression of Kx antigen,

weak expression of all Kell system antigens and
Late-onset muscular, neurological and psychiatric disorders,
acanthocytosis of RBCs in 30 %, with haemolytic anaemia.

In some cases, an X chromosome deletion is large enough to encompass XK and

CYBB, a gene for a subunit of flavocytochrome b558, which leads to McLeod
syndrome and chronic granulomatous disease (CGD).
 THE ANTIGLOBULIN TEST
 Antiglobulin serum (Coombs’ Serum) was discovered by
Coombs in 1945.
 The antiglobulin test can be used to detect red cells sensitized

with IgG alloantibodies,

alloantibodies IgG autoantibodies or complement
components.
components
 Sensitization of red cells can occur in vivo or vitro.

 The use of AHG serum to detect sensitization of red cells in

vitro can be:

 One stage technique , the direct antiglobulin test (DAT).
 Two stage technique , the indirect antiglobulin test (IAT).
 PRINCIPLE
 Normal human red blood cells, in presence of antibody
directed towards the antigen they possess, may fail to
agglutinate when centrifuged and become sensitized.
sensitized This may
be due to the particular nature of the antigen and antibody
involved.
 Sensitization of RBC’s may be with IgG or complement.

 In order for agglutination to occur an additional of anti-

antibody or anti-complements, which reacts with the Fc portion

of the IgG antibody, or with the C3b or C3d component of
complement alternatively.
PRINCIPLE

 This will form a "bridge" between the antibodies or

complement coating the red cells, causing agglutination.
 The coating (sensitization) of red cells can occur in vivo or in
vitro following incubation at 37°C with serum containing
antibody.
 PRODUCTION METHODS OF ANTI-HUMAN
GLOBULIN (AHG OR COOMBS) REAGENT
 May be made by injecting rabbits , goats or sheep with purified human IgG
or C3, then harvesting the antibodies produced by the rabbit.
 Monoclonal technology may be used to make monoclonal antiglobulin
reagent.

TYPES OF AHG REAGENTS

Polyspecific Anti-human Globulin: blend of Anti-IgG and Anti-C3b, -C3d
Monospecific reagents: Anti-IgG alone or Anti-C3b,-C3d alone

Note:  Reagent does not contain antibodies to IgM.  Information about

IgM coating of cells comes from the presence of C3 coating the cells
since IgM is a strong complement activator.
Polyclonal or monoclonal
sources :
Polyclonal - animals
hyperimmunized with
human globulins and bled
bled for a production
batch of reagent for
antisera to obtain high
titered, high specificity to
human
Monoclonal - hybridoma cells from mice as follows:
Mice hyperimmunized with human globulins
Prepare cell suspension from spleens; fuse with immortalized myeloma cells
Screen hybridoma clones for desirable specificities and affinities
Maintain cultures of clones in vivo or in vitro
TYPES OF AHG REAGENT
 Polyspecific Anti-human Globulin: blend of Anti-IgG and Anti-
C3b, -C3d
 Monospecific reagents: Anti-IgG alone or Anti-C3b,-C3d alone

Note:  Reagent does not contain antibodies to IgM. 

Information about IgM coating of cells comes from the
presence of C3 coating the cells since IgM is a strong
complement activator.
 Detects sensitized red cells with IgG and/or complement components C3b
and C3d in vivo.
 In vivo coating of red cells with IgG and/or complement may occur in any
immune mechanism is attacking the patient's own RBC's. These mechanism
could be:
 Autoimmunity
DAT
 Alloimmunity
 Or a drug-induced immune-mediated mechanism.
 EXAMPLES OF ALLOIMMUNE
HEMOLYSIS
 Hemolytic transfusion reaction
 Hemolytic disease of the fetus & newborn (also known as
HDN or erythroblastosis fetalis)
 Rhesus D hemolytic disease of the newborn (also known as Rh
disease)
 ABO hemolytic disease of the newborn (the indirect Coombs test
may only be weakly positive)
 Anti-Kell hemolytic disease of the newborn
 Rhesus c, E hemolytic disease of the newborn
 Other blood group incompatibility (RhC, Rhe, Kidd, Duffy, MN, P
and others)
HEMOLYSIS
 Warm antibody autoimmune hemolytic anemia
 Idiopathic
 Systemic lupus erythematosus
 Evans' syndrome (antiplatelet antibodies and hemolytic
antibodies)
 Cold antibody autoimmune hemolytic anemia
 Idiopathic cold hemagglutinin syndrome
 Infectious mononucleosis
 Paroxysmal cold hemoglobinuria (rare)

Drug-induced immune-mediated hemolysis

Methyldopa (IgG mediated type II hypersensitivity)
Penicillin (high dose)
Quinidine (IgM mediated activation of classical complement pathway and Membrane
attack complex)
  Whole Blood Sample - It should be as fresh as
Blood Sample
possible not more than 24 hours old,
 otherwise, the sample should be taken in
EDTA.
PROCEDURE OF DAT
1. Take 2-3 drops of blood to be tested in a clean labeled tube.

2. Wash the red cells 3-4 times in a large volume of saline to remove free globulin
molecules. Remove all supernatant after each wash. Completely decant the final
supernatant wash.

3. Add 2 drops of polyspecific AHG serum in 1 drop of sensitized washed red cells or in
1 drop of 3-5 % suspension of sensitized cells immediately.

4. Mix, Centrifuge at 1000 rpm for 1 minutes immediately.

5. Gently shake the tube to dislodge the cell button and see for agglutination, use optical
aid if needed, Record the result.

6. Add 1 drop of IgG coated red cells to a negative test. Mix, centrifuge at 1000 rpm for
1 min. Immediately look for agglutination. If a negative result (no agglutination) is
obtained the test result is invalid and whole test should be repeated. If agglutination is
obtained, the result is valid.
(IAT)
Indications
 to determine the presence of sensitization of red cells with IgG and/or
complement in vitro in the following conditions:
1. Compatibility testing.
2. Screening and detection of unexpected antibodies in serum.
3. Determination of red cells phenotype K, Lea, Fya Fyb, Jka, Jkb and
sub-group of Rh etc by using known sera.
 :PROCEDURE
1. Place 2-3 drops of the test serum in a tube. Serum should be
fresh for detecting complement components and complement
binding antibodies, otherwise, fresh AB serum should be
added to it.
2. Add 1 drop of 3-5% suspension of washed O Rh (D) positive
red cells to the serum in the tube.
3. Mix and incubate at 37°C for 30-40 minutes.
4. Centrifuge at 1000 rpm for 1 minutes.
5. Examine for hemolysis and/or agglutination. Use optical aid if
necessary. Agglutination at this stage indicates the presence of
saline (complete) antibodies.
6. If no agglutination is seen, wash cells 3-4 times in large
volume of saline. Decant supernatant in each wash as
completely as possible.
PROCEDURE:

7. Add 2 drops of AHG serum to the cells.

8. Mix and centrifuge at 1000 rpm for 1 minutes immediately.
9. Gently shake the tube to dislodge the button and examine for
agglutination, using optical aid. Record the result.
10. Add 1 drop of IgG coated red cells to any test that is
negative. Mix and centrifuge at 1000 rpm for 1 minutes.
Look for agglutination. If there is no agglutination, the test
result is invalid and the whole test is repeated. If
agglutination is obtained the result is valid.
11. Auto control should be kept with IAT.
BOVINE ALBUMIN(22%)-IAT
ONE STAGE METHOD - ADDITIVE METHOD
 Procedure:
1. Two drops of albumin
22.5% are added in
step (2) of saline-IAT
2. Mix and incubate for
20-30 minutes at 37°C
3. Proceed further as in
saline-IAT procedure.
SOURCES OF ERROR IN AHG TESTS
False negative results: General DAT & IAT
Failure to wash red blood cells adequately, since globulins not bound
to RBCs will neutralize the AHG reagent.
 The washing process and the addition of AHG reagent must be
undertaken as quickly as possible to minimize loss of bound antibodies
by elution.
Improper storage, bacterial contamination and contamination with
human serum will impair the AHG reagent activity.
Not adding the AHG reagent

Improper centrifugation

Number of cells present in the test:

 too many cells give weak reactions
 too few cells will impair the reading of the agglutination
FALSE NEGATIVE
RESULTS
 DAT
 All samples negative at the AHG phase should be incubated at
room temperature for 5 minutes to achieve maximal sensitivity
needed for complement detection.
 IAT
 Serum and/or rbcs lose reactivity if improperly stored.
 Plasma used instead of serum can lead to failure to detect
antibodies depending on presence of active complement (anti-Jka,
-Jkb)
 Temperature and incubation time affect attachment of antibody or
complement to cells.
 An optimal proportion of serum to cells should be achieved:
usually 2-3 drops serum to one drop of 5% cell suspension.
:FALSE POSITIVE RESULTS

 DAT and IAT ;

 In specimens containing potent cold-reactive antibodies
agglutination may occur before adding the AHG reagent.
 Dirty glassware may cause clumping of cells.
 Over centrifugation
 DAT
A positive DAT from a clotted sample should be repeated on an
EDTA sample
 Samples collected from infusion lines may have complement
present on the cells.
 IAT
 Cells with a positive DAT will give a positive result in any indirect
antiglobulin procedure.
 COOMB’S CELLS
 To show that test cells were properly washed and that no neutralization
or reagent deterioration has occurred, antibody-coated cells are used
as a positive indicator.
indicator
 In a negative antiglobulin test the anti-human globulin should remain

active and this can be demonstrated by the addition of IgG sensitized

cells.
 Agglutination of the IgG sensitized cells after mixing and centrifuging

confirms that the anti-human globulin was added to the test, that the
test cells were properly washed and all free globulin molecules were
removed and that the anti-human globulin was active.
 Failure of the IgG sensitized cells to agglutinate indicates that the

original negative antiglobulin test result is not valid and testing must
be repeated.
 PREPARATION OF COOMB’S
CELLS

 Very easy: To about 10 drops of washed O Positive red cells

add 5-6 drops of anti-D antisera. Incubate at 37C for 15
minutes. Wash 4 times then prepare a 3 to 5% cell suspension.
 To verify reaction, add two drops of AHG into test tube and
one drop of newly prepared Coombs cells.
 Centrifuge on High speed for 15 seconds , You should get 1-2
+ reaction.
BLOOD COMPONENTS
& PLASMA DERIVATIVES

112
Whole Blood (WB)
• Collected directly from donors into blood
transfusion bag containing anticoagulant
• 500 ml transfusion bag is used (contains
63 ml of anticoagulant + 450 ml blood)
Whole blood is indicated when massive
transfusion is needed :
a- when there is a deficit O2 carrying
capacity ; ex. Shock , Thalassaemia
b- massive hypovolaemia (bleeding) 113
c – Cardiac and arterial surgery .
 Anticoagulants in blood units
1) Acid-Citrate-Dextrose (ACD)
2) Citrate-Phosphate- Dextrose (CPD)
3) Citrate,Phosphate,Dextrose,Adenine (CPDA-1)

Additive solutions

• (SAGM) → Saline-Adenine-Glucose- Manitol

• Purpose of additive solution, to improve RBCs storage viability till 42
days @ 2-6ºC

* Added only to PRBCs 114

Anticoagulants Used for WB?
• ACD & CPD preserve the unit for 21 days at 2-6ºC.
• CPDA-1 (anticoagulant/preservative for 35 days).
• C = Citrate→ to prevent clotting
• P = Phosphate→ to maintain pH
• D = Dextrose→ ATP generation
• A = Adenine-1→ substrate from which RBC produce ATP

Anticoagulant ratio is 1.4 ml:10ml blood (63ml / 450ml) 115

BLOOD COMPONENTS

• Human blood consists of

plasma, in which cells are
suspended

116
• The plasma also contains
other specialised substances,
which are important for blood
clot formation (e.g. clotting
factors)
• Whole blood can be separated
at the blood bank into various
components
BLOOD COMPONENTS
 Blood separated into different parts:
1. Packed red cells
2. Platelets

117
3. Fresh frozen plasma
4. Cryoprecipitate
5. Granulocytes
6. Factor IX conc.
7. Factor VIII conc.

 There are more than 20 different products

available
 Whole blood

Red cells Granulocytes Plasma Platelets

Fractionated
frozen )Fresh(
products
plasma (FFP)

F Vlla -Immuno
globulins

F Vlll
Stored Albumin
Cryoprecipitate
Plasma

F lX
118
BLOOD
COMPONENTS

 Refers to a product separated from a single unit

of whole blood

119
 The term plasma derivative indicates a blood

product separated from a large volume of pooled

plasma by a process called fractionation

Blood Components Separation Goals

• Decrease harmful effects of blood transfusion.

• Giving patients specific component needed.

• Allow a longer survival for components.

• More than one patient will use the unit(so,

avoid wasting of units). 120

 Centrifugation Types?

There are two types of centrifugation:-

• Light spin; (2000 rpm at 20ºC for 11 min)

• Heavy spin; (3500 rpm at 20ºC for 11 min)

121
 Top and bottom
(buffy coat) method
Centrifuged
blood

Plasma

Buffy Coat
(WBCs & Platelets)

Red Blood Cells

122
 BLOOD COMPONENTS
 Blood components • Plasma Derivatives
 Oxygen carrying components – Coagulation Factor
 Red cell concentrates concentrates
(RCC) • Factor VIII concentrates
 Leukocyte poor blood • Factor IX complex

123
 Frozen-thawed red cells concentrates & others
 Platelet products – Oncotic agents
 Platelet rich plasma (PRP) • Albumin
 Platelet concentrates (PC) • Plasma protein fraction
 Plasma products (PPF)
 Fresh frozen plasma (FFP) – Immune serum Globulin
 Frozen plasma (FP) • Hepatitis B Ig (HBIG)
 Cryoprecipitate • Varicella-zoster Ig (VZIG)
 Stored plasma • Rh Ig (RhIG)
• Tetanus Ig (TIG)
A- BLOOD COMPONENTS THAT CARRY
OXYGEN

 Increase the oxygen carrying capacity of the blood by

increasing the circulating red blood cell mass.

124
 Carry oxygen and nourishment to the tissues and take
away carbon dioxide.
PRBCs -1
How to make (PRBCs)?
 RBCs have higher specific gravity than
plasma, it moves to lower portion of the bag
by centrifugation
 WB (Light spin) Two products:
1) PRBCs
2) Platelet Rich Plasma (PRP)

125
Whole Blood Unit

After centrifugation
WB separates into
plasma & platelets &
PRBCS

126
1 2 3

SAGM

127
RED BLOOD CELL -1
CONCENTRATES
 Prepared by removing approx.
200 ml of plasma from whole

128
blood after centrifugation
 RBCs plus 100 ml of residual
plasma
 In CPD-A can be stored for 35
days at 4oC
RED BLOOD CELL CONCENTRATES -1
Red cell Whole Blood
concentrate
300 ml 500 ml Total Volume

200 ml 200 ml Volume of

red cells
100 ml 300 ml Volume of
plasma
70 % 40 % Hematocrit
129
RED BLOOD CELL -1
CONCENTRATES
 High hematocrit → viscous → infuse slowly
 Rate of infusion increased by adding saline
 Other fluids should not used

130
 Calcium containing fluids (eg. Ringer’s lactate) should
not be added
 May cause clotting
 Glucose solutions
 can cause clumping
 Only saline can be added to blood
24 hrs
Expiration date!

Once the PRBC unit is “opened” it has a 24 hour expiration date!

 2- LEUKOCYTE POOR
BLOOD
 No viable leukocytes Buffy coat
 WBCs are of no Red cells
consequence
 In some patients with H/O
febrile transfusion reaction,

131
should receive leukocytes
poor-blood
 WBCs can be removed by:
 discarding the buffy coat
(inverted
centrifugation/hanging)
 washing RBCs
 using leucocyte-depleting
filters(maintain closed
system
Final unit must have < 5 x 106
WBCs
FROZEN-THAWED RED -3
CELLS
 Red cells can be frozen with use of cryo-preservation techniques
 Permit storage for up to 10 years

 Expensive procedure & recommended only in special

circumstances

132
 e.g.Individuals with rare blood types
 For auto-transfusion
 The RBC's are first incubated in a 40% glycerol solution which
acts as an "antifreeze" within the cells.
 The units are then placed in special sterile containers in a deep
freezer at less than -60 degrees C.
 Cryopreserved units are thawed and washed free of glycerol prior
to use as saline suspended RBC's.
FROZEN-THAWED RED CELLS -3
 Deglycerolized RBCs
 RBCs that have had the glycerin
removed
 Thawed at 37°C
 A blood cell processor washes the
cells with varying concentrations
of saline
 Considered “open”, expires in 24
hrs.

4. Washed RBCs
Not effective in reducing WBCs
For patients (with anti-IgA) that may react with plasma proteins
containing IgA 133

Reactions may be allergic, febrile, or anaphylactic

IRRADIATED RBCS -5
 Irradiated RBCs
 Prevents T-cell proliferationthat
may cause transfusion-associated
graft versus host disease (GVHD)
 GVHD is fatal in 90% of those
affected
 Used for:
 Donor units from a blood relative
 HLA-matched donor unit

 Intrauterine transfusion

 Immunodeficiency

 Premature newborns

 Chemotherapy and irradiation

 Patients who received marrow or stem

cells
134
SYNTHETIC OXYGEN CARRYING -6
AGENTS
 Synthetic oxygen carrying agents
 Perfluorochemical (e.g. Fluosol-DA )
 Fluorinated hydrocarbons
 Readily dissolve oxygen

135
 Poor soluble in plasma

 Side effects:

 Hypotension
 DIC

 Chemically modified hemoglobin

 Free Hb has a very short half life
 Chemically modified to:

 increase intravascular survival

 and to make it more effective in carrying oxygen
B- Platelets Important in maintaining hemostasis
Help stop bleeding and form a platelet plug
(primary hemostasis)
People who need platelets:
Cancer patients
Bone marrow recipients
Postoperative bleeding

HOW PLATELETS ARE PROCESSED

 REMEMBER!!!
 Requires 2 spins:
 Soft– separates RBCs and WBCs from plasma and platelets
 Heavy
 platelets in platelet rich plasma (PRP) will be forced to
the bottom of a satellite bag
 40-60 mL of plasma is expelled into another satellite bag,

while the remaining bag contains platelet concentrate 136

PREPARATION OF PLATELET
CONCENTRATE

Plasma

RBCs PRP

Platelet
concentrate 137
 B- PLATELET PRODUCTS
 Platelet Rich Plasma (PRP)
 Gentlecentrifugation of whole blood
 Supernatant transferred to the 2nd bag
 Platelet Concentrates
 Prepared from PRP by a 2nd centrifugation

138
 Removal of all but 50 ml of plasma
 Contain approx. 6X1010 platelets
 60 – 80% Plts present in whole blood unit
 Remain 5 days
 Longer at 22oC with continuous agitation

Contamination by WBCs & RBCs is usually small But there is enough to induce
alloimmunization.
Plt concentrates from Rh +ve should not be administered to Rh –ve women.
Storage at 22oC, therefore care to prevent contamination
 2
1

Then

139
 Whole
Wholeblood
bloodunit
unit

Centrifuge using LIGHT spin

Express Platelets Rich Plasma (PRP) into satellite bag

Take PRP and centrifuge again now using HEAVY spin

Express PPP into satellite bag & freeze at -18ºC

140

Final products :PRBCs, Platelets concentrate, FFP

2- Platelets Concentrate (PC)
How to prepare PC?
Platelet Rich Plasma (PRP)
centrifuged using (heavy spin),
this will produce:
1) Fresh frozen plasma (FFP)
2) Platelets concentrate (PC)
• PC are stored at room temperature on
platelet agitator (prevent platelets clumping)
• PC stored for 5 days at 20-24°C.
141
• Each unit should elevate the platelet count by
5000/µL
 Platelets concentrate -2
• Indications:
1.To prevent bleeding due to
thrombocytopenia or platelet dysfunction
2.To a patient undergoing an operation, if
the platelet count is less than 20,000/µL

142
1 2 3

143
1- Fresh frozen plasma (FFP)

Freeze at -18ºC for 1 year from collection date.

Or freeze at -70ºC for up to 7 yrs
Cross match is not required, but of coarse should be ABO
compatible.
• Liver disease
• Severe burns
• Provides coagulation factors for
Indications – Bleeding
Of – Abnormal clotting due to massive transfusion
– Patients on warfarin who are bleeding
FFP – Treatment of TTP and HUS
– Factor deficiencies 144
– ATIII deficiency
– DIC when fibrinogen is <100 mg/dL
 Plasma Products
Plt-poor plasma can be separated into a number of products
Fresh frozen plasma
Frozen plasma
Cryoprecipitate
Stored plasma
FRESH FROZEN PLASMA -1

145
(FFP)
 Prepared from whole blood within 6
hours of collection
 Rapid freezing of plasma preserves
the labile coagulation factors at
maximum levels
 Don't contain cellular elements

 200 ml volume
FROZEN PLASMA -3
(FP)
 Separated from whole blood within 24 hours of
collection
 Contains at least 50 % of original factor VIII & factor V
frozen plasma

146
 Adequate source for treatment of mild to moderate
coagulation factor deficiencies
 200 ml volume
 Storage at -30oC for up to 12 months
CRYOPRECIPITATE
 Produced from freshly separated plasma by freezing at -70oC
followed by thawing at 4oC
 It is rich in factor VIII, fibrinogen and fibronectin
 Once thawed, mixture is centrifuged to sediment the cryoprecipitate
& all but 5 to 10 ml of supernatant plasma is removed
Indications:

147
Hemophilia A
Von Willebrand disease (VWD)
 Contains 250 mg fibrinogen Congenital or acquired fibrinogen defects
(i.e., dysfibrinogenemia)
 80 clotting units of factor VIII
 Stored at -30oC for 12 months
 Increase of 2% of factor VIII level for each bag of cryoprecipitate
infused
 Supernatant plasma removed is called stored plasma
 Must be used within 5 weeks if stored at 4oC
 Lasts for 2 years at -30oC
5- STORED
PLASMA
 Plasma separated from whole blood after 24 hours of storage at
4oC
 Can also be derived from cryoprecipitate production

 Contain reduced levels of labile coagulation factors V VIII &

148
fibrinogen
 It is indicated for patients requiring volume expansion or
protein replacement when labile clotting factors are not required

 Plasma products do not require crossmatch prior to use but

should be ABO compatible
Summary:Blood Components
Indication Storage Centrifugation Blood Component
Temp Time

•Anemia 2-6ºC +SAGM 42d WB Light spin= 1) PRBCs

•Newborn 2000rpm-20ºC -11min.
exchange PRBCs + PRP
transfusion
•Bleeding R.T 3-5 d PRP heavy spin= 2) PC
•Operation if plt. 3500rpm-20ºC -11min.
Less than PC + FFP
20000/μl
•Clotting factor -18ºC 1year 3) FFP
deficiencies -65ºC 7 years
•Severe burns
•Hemophilia A 1 year -30ºC a. WB special heavy 4) Cryo
• Von Willebrand spin= 3500rpm at
disease 4ºC -11min.
RBC + Plasma
b. Plasma store
at -18 ºC then 149
thaw at 4 ºC then
heavy spin at 4ºC
PLASMA DERIVATIVES
 Certain plasma derivatives can be obtained by fractionating the fresh frozen
plasma or stored plasma
 Fractionation:
 Allows the processing of large volumes of pooled plasma
 Pooling of many units increases the risk of viral transmission to the

150
recipient
Preparation avaliable Plasma Derivatives
Factor VIII concentrates
Factor IX concentrates Coagulation Factors
Anti-thrombin III
Albumin
Albumin
Plasma protein fraction
Non-specific immune serum globulin (ISG)
Rh immune globulin (RhIG)
Hepatitis B immune globulin (HBIG) Immune globulins
Varicella-Zoster immune globulin (VZIG)
Tetanus immune globulin (TIG)
PLASMA PROTEIN
FRACTIONATION
 Plasma proteins are separated according to differences of each
protein.
 Fractionation involves changing the conditions of the pooled
plasma (e.g. the temperature or the acidity)
 Proteins that are normally dissolved in the plasma fluid

151
become insoluble, forming large clumps, called precipitate.
 The insoluble protein can be collected by centrifugation.

One of the very effective ways for carrying out this process is the
addition of alcohol to the plasma pool while simultaneously cooling the
pool.
This process is sometimes called cold alcohol fractionation or
ethanol fractionation.
This procedure is carried out in a series of steps so that a single
pool of plasma yields several different protein products, such as
albumin and immune globulin.
COAGULATION FACTOR -1
CONCENTRATES
 Prepared in a freeze-dried form
 Indicated for patients with congenital coagulation deficiencies
 Risk of hepatitis is high
 Should not used for mild acquired

152
coagulation deficiencies
 Should be treated with FP or FFP

Factor VIII Concentrate

Commercially prepared, lyophilized powder purified from
human FFP
Contain also small amounts of fibrinogen & other proteins
Can contain blood group Abs
Treat patients with hemophilia A
DIFFERENCES OF CRYOPRECIPITATE & FACTOR VIII
CONCENTRATES

Factor VIII
Cryoprecipitate
concentrates
4oC
-30oC Storage Temp.
Short period RT
Risk of
High Low
Hepatitis
Treatment of
Yes Yes
hemophilia A
Treatment of
no Yes 153
vW disease
 FACTOR IX
CONCENTRATE
 For the treatment Factor IX deficiency or Hemophilia B (Christmas
Disease).
 Have been used to treat patients with acquired inhibitors of factor
VIII

154
 Have factor VIII bypassing activity
 Contains also factors II, VII & X in concentrated form

 Vials containing 500 units of factor IX

 It is contraindicated in patients with liver disease

 Have low levels of circulating antithrombin III

 Activation of clotting factors present in some factor IX
concentrates,
 cause DIC
BLOOD PRODUCTS & TREATMENT OF SPECIFIC CLOTTING FACTOR
DEFICIENCIES

Blood product Indicated Deficiency

Cryoprecipitate
Fibrinogen
Stored plasma
Fresh frozen plasma
Factor V
Frozen plasma
Factor IX concentrate
Factor VII
Stored plasma
Factor VIII concentrate
Factor VIII
Cryoprecipitate
Cryoprecipitate
Fresh frozen plasma Von Willebrand’s Disease
Frozen plasma
Factor IX concentrate Factor IX
Stored plasma Factor X
Stored plasma Factor XI 155
Stored plasma Factor XIII
 Albumin: volume expansion
Other colloids are available for blood
volume expansion
2- Oncotic Agents Dextran
Gelatin
Hydroxyethyl starch
ALBUMI Polyvinylpyrrolidone

156
 Albumin is prepared by ethanol fractionation of pooled plasma
 Available in 5% and 25% concentrations.
 Have physiological sodium content
 No risk of hepatitis, sterilized during preparation
 No coagulation factors or blood group Abs
 Used for treatment of hypovolaemia and hypoalbuminaemia (result from
abnormal synthesis, increased metabolism or loss)
 It maintains capillary osmotic pressure
 Carrier protein for drugs, hormones, enzymes & metabolites
 PLASMA PROTEIN
FRACTION
 Partially purified albumin
 Contains ≈ 85% albumin & 15% other plasma proteins

Immune Globulins -3

157
Contains immune IgG antibodies, prepared from pools of
plasma.
For disease prophylaxis, hepatitis A, measles, varicella and
rubella.
For the treatment of hypogammaglobulin-emia and
agammaglobulinemia.
IMMUNE SERUM GLOBULIN
(ISG)
 Primarily IgG Ab
 Prevention of some viral diseases

 Hypogammaglobulinemia

 Congenital immune deficiency

158
 Given by IM injection (aggregates of IgG)

Hepatitis B Immune Globulin (HBIG)

Contains Hepatitis B immune antibodies.
From plasma of donors with high titer of Ab to HBsAg
Provides passive immunization for HBV.
For treatment after exposure to HBsAg.
For the prevention of maternally transferred HBV (perinatal exposure).
VARICELLA-ZOSTER IMMUNE GLOBULIN
(VZIG)
 Derived from patients had recent Herpes Zoster infections
 Herpes Zoster infections result in severe fatal infection in
immunocompromised individuals
 Passive administration of VZIG during 72 hours of exposure can

159
prevent or attenuate infection

Tetanus Immune Globulin (TIG)

Prepared from individuals specifically immunized
for tetanus toxoid
Available for individuals at risk following injury
RH IMMUNE GLOBULIN
(RHIG)
 Derived from Rh -ve individuals
 Contains IgG antibodies to the D antigen on red blood
cells.
 Given during pregnancy and post-natally to Rh

160
negative mothers to prevent the development of anti-D
and hemolytic disease of the newborn (HDN) due to
anti-D.
 Given prophylacticaly following abortion, or invasive
maternal procedures (e.g., amniocentesis).
Changes in stored blood:

Certain percent of RBC are destroyed, in good

anticoagulant only 10-20 % of RBC are destroyed.
Blood become acidic.
Elevation in K concentration which is bad for heart.
Platelets will die within few hours after collection.
So there will no Plts.
WBC are deteriorated also (no WBC in old blood).
Activity of coagulation factors will be grossly
reduced (VIII, V). 161
GRANULOCYTES
CONCENTRATE
 Neutrophils are the most numerous, involved in phagocytosis of
bacteria/fungi
 Although rare, it is useful for infants with bacteremia
 Prepared by hemapheresis
 ≥ 1.0 x 1010
 Maintained at room temp for 24 hours

162
 BLOOD DONATION
According to WHO regulation: should be a voluntary process and charge-free
 Principles of blood donar selection :

1. The donor should be in a state of good health .

2. A Consent : should be signed, for legal purposes

3. Age : restricted to adults ; range 18-65 years .

4. The donor temp. should not > 37.5 0 C or 99.5 0 F

5. Weight : not < 50 kg .(110 Ib) ;
Wt of donar (kg) x 450
50
6. Hemoglobin level : Minimum: 13.5 g/dl(PCV 41.0 %) for males, 12.5 g/dl (PCV
37 %) for females

7. Minimum allowable time between blood donations : 12-16 weeks

Patients on hemodialysis deferred from blood donation .
A history of jaundice should be assumed to be due to hepatitis unless proved otherwise and should be
tested for HBs Ag and anti-HCV.
Donor conditions of primary concern to the recipient .
Blood transmitted diseases including :
Hepatitis :
Those who had history of viral hepatitis or of + HBs : deferred for 12 months if HBs Ag testing is
negative .
Hepatitis A virus : rarely transmitted by transfusion .
close contact with hepatitis patients: should be deferred as blood donors for at least 6 months

At higher risk of hepatitis

1. Narcotic users .
2. Prison inmates
3. Tattooing: deferred for at least 6 months.
4. Acupuncture and ear piercing
Human immunodeficiency virus
( HIV - l and HIV - 2).
can be transmitted in both cellular
and plasma components
Deferred for life
Certain behavior patterns place
individuals at greater risk of HIV
infection.
 male homosexuals and bisexuals,

 intravenous drug users

 prostitutes
 Hemophiliacs treated with blood
products permanently
 The sexual partners of such deferred from blood
individuals donation.
Cytomegalovirus
 post-transfusion infectious mononucleosis-like syndrome (especially
after the transfusion of large amounts of blood).
characterized by:
 fever,
 splenomegaly
 atypical lymphoid cells in the peripheral blood,
 negative Paul Bunnell test.

The groups at particular risk:

 premature babies weighing less than 1500 g,
 bone marrow and other organ transplant recipients,
 pregnant women (the fetus is at risk).
 Immunosuppressed patients

All antibody-positive donors should be considered as having the potential

to transmit CMV.
As CMV is WBC-associated, effective leuco-depletion should provide
similar levels of safety as serological testing.
Other viral diseases :
Donor expose to measles , Germen measles , chickenpox or mumps by family
contact wait one incubation period ( about 3 weeks) before accepting the
donor.
Infectious mononucleosis can be transmitted by blood transfusion and sometimes
it is difficult to differentiate it from infectious hepatitis. Since patient with IM
may suffer from lethargy for several months therefore it is better to defer them
for at least 6 months after the symptoms have subsided .

Babesiosis: It is an intra-erythrocytic protozoan parasite and it is spread by

tick bites and commonly afflicting cattle or large rodents. Human infection
usually caused by B. microti producing an acute febrile illness that may be
fatal. In endemic areas it is advisable to defer donor with unexplained fever or
recent tick bites.

Tuberculosis :
As long as the donor has no symptoms and is not under therapy, he or she can
give blood . In rare circumstances it can be transmitted by blood transfusion.
Syphilis
Treponema pallidum does not survive well at 4 ° C and red cell preparations are
likely to be non-infective after 4 days ’ refrigeration
more likely to be transmitted in platelet concentrates, due to their room
temperature storage and short shelf - life.
Donor with positive VDRAL, TPHA: permanently debarred.
Malaria
Malarial parasites remain viable in blood stored at 4 ° C and are readily
transmissible by blood transfusion .
In the UK, donors with a history of possible malaria exposure (provided it was
more than 6 months previously and they are free of symptoms) can be accepted
if they are negative by ELISA for malarial antibodies.
Bacteria
The vast majority of Bacterial transmissions are due to contaminated platelet
preparations that are more than 3 days old, because bacteria (mostly skin
commensals) proliferate easily at room temperature.
Prions
Variant Creutzfeldt – Jakob disease (vCJD), the human form of bovine spongiform
encephalopathy (BSE), is considered a potential threat to blood safety. Appropriate
donor exclusions and the introduction of leuco-depletion of all blood components are
an important precautionary interventions.
Inoculations and injections :
In those receiving cell -free vaccines or those containing only killed organisms(Salk
poliomyelitis vaccine and inoculation for hepatitis B V, typhoid , typhus , Rocky
mountain spotted fever , cholera , plague , diphtheria .pertussis , influenza , rabies
and tetanus) , can donate at any time after immunization providing they feel well .
In live virus vaccines(oral poliomyelitis vaccine, measles (rubeola) , mumps , yellow
fever and small pox ), it is recommended that a two weeks waiting period after
inoculation since donor viremia in immunosuppressed patients may cause disease
.
Longer deferments are adviced for Germen measles (rubella) , which require waiting
for 1 month to abolish the chance of transmitting the virus to pregnant recipient .
Also for rabies it is adviced to defer the donation of blood for 1 year ,since rabies may
have incubation period of several months .
Patients who received Hepatitis B immune globulin should be deferred for 1 year .
Miscellaneous Donor conditions :
Hereditary red cell defect:
G-6-PD deficiency
thalassaemia minor may be accepted to donate
sickle trait their blood may create difficulty in freezing ,
thawing procedure or rarely subject to
Allergies: Allergy may be transferred from donor
hemolysis aftertotransfusion.
recipient by means of plasma and
a recipient may experience allergic symptoms on exposure to the corresponding
allergen . Seasonal allergy is benign and may be disregard .
Allergy in the donor to drug and food are more serious than hay fever.
Donor on antihistamines may be accepted since transmission of significant amount of
the drug is unlikely .
Dental Surgery: Should be deferred for 3 days to allow time for recovery since there is
chance for bacteremia after such procedure .
Skin disease:
Minor skin lesion such as acne , psoriasis and seborrheic dermatitis: can donate
Patients taking isotretinoin (13-cis-retinoic acid or Accutane ) for cystic
acne(teratogenic agent and presents a risk to the fetus if transfused to pregnant
mother): deferred from blood donation and can give blood after at least 1 month of
stopping the drug.

Conditions Affecting the donar’s response to phlebotomy :

Heart disease
Hypotension and bradycardia such as occur after about 1% of blood donation may be
dangerous to any one with narrowed coronary vessels and may lead to myocardial
infarction because of poor perfusion of the coronary arteries.
The donor should be asked if he had previous coronary heart disease , heart attacks ,
angina pectoris , shortness of breath and other sign and symptom of heart
problems .Also patient who underwent cardiac surgery or those who had
cerebrovascular accident .
Rheumatic fever and Rheumatic heart disease : Donor should be free of all evidence of
rheumatic heart disease . As well as patient who is under treatment for rheumatic
heart disease as it is likely that there might be recurrence of the disease after
donation of blood .
Abnormalities of pulse and blood pressure :
pulse should be regular and 50-100 beat / min
Normal BP for age
: Recent and major surgery deferred for at least 6 months
Diabetes Mellitus :
Deferred, esp a severely diabetic patient, on medication such as insulin and
tolbutamide.
Epilepsy or fit : permanently disqualified
Blood diseases: polycythemia rubra vera unfit,
Secondary polycythemic can donate.
All patients should be questioned about bleeding tendency otherwise the donor
may bleed form the venipuncture .
Medication taken by the donors :
In general the amount of drug in a unit of blood is negligible although it could be
enough to cause reaction in an allergic recipient .
Patients on isotretinoin should be deferred from blood donation and can give
blood after at least 1 month of stopping the drug .
Aspirin had an advert effect on platelet function but this effect is negligible in a
unit of blood because of the presence of enough un-aspirinated platelet .
However when platelet is collected by platelet apheresis , it is recommended by
the FDA that aspirin should be stopped for 36 hours whereas the AABB has a
stricter requirement of 3 days .
Some hypertensive patients taking antihypertensive drug should be deferred even
if BP is normal , since they have some circulatory instability and their response
to a vasovagal reaction is unpredictable .
Donor ‘s Occupation :
Those expected to return immediately to a hazardous activity, or scafolders, crane
workers: unfit

Pregnancy: deferred for at least 6 weeks after delivery or termination of

pregnancy .
Menstruating female need not be deferred as long as they feel well and there is no
excessive flow .

Blood collection :
Blood drawing Area : This area should be pleasant , clean , quiet and separated
from other parts of the blood bank . Soft background music helps relax some
blood donor .
Venipuncture Site : A large vein in the ante cubital region
Pressure applied to the upper arm by means of a blood pressure cuff just about the
diastolic pressure ,
the donor clinches his fist.
The chosen area should be cleaned thoroughly to ensure asepsis.
Phlebotomy:
A large 15-17 gauge needle is used and remain in the vein for 4-7 min., maintaining
free blood flow the entire time .
The blood container must be kept below the level of the needle in the arm , to facilitate
the mixing of blood with anticoagulant , the container should be in an inverted
position during collection and agitating the container by a mechanical agitator
which is provided with a weight balance calibrated to shut off the blood flow when
the proper amount of blood has been collected. The donor tubing should be clamped
before the needle is removed or the tubing cut .
Enough donor tubing is left attached to the bag to allow preparation of sufficient no.
of segments . The segments remain connected to the pint and to each other and are
used for cross match .
After the removal of the needle the donor raises the arm and applies gentle pressure
;and the donor should be observed for at least 10 min, and any abrupt change of
position should be avoided because it may cause dizziness.
Smoking or alcohol intake soon after donation must be prohibited because may cause
syncopal reaction .
Adverse reaction to blood donation :
Fainting (Syncope) :
The most frequent reaction is simple faint.
caused by a neurophysiological response to blood loss
precipitated by the sight of blood .
more common in first time donors(because of fear).
feeling of dizziness or light headedness
tingling of the finger
cold, clammy feeling in the palms of the hands .
The donor ‘s face appears almost greenish pale, with cold sweaty palms and beads
of sweat appear first on the upper lip.
loss of consciousness for few moments or longer .
Low BP
bradycardia.
Reaction variation:
1. In 1/3rd : severe reaction + vomiting attack .
2. 1/3rd : neuromuscular excitability, usually associated with hyperventilation 
carpopedal spasm and stiffness of the face and lip. Occasionally this may lead to
more generalized convulsive movements .
3. 1/3rd delayed syncopal reaction in which fainting may occur as late as 30 min. to
hour after donation , by which time the donor had left the blood bank .The
donor may fall and be injured and it may be dangerous if the donor is engaged
in some hazardous occupation .

Donors are advised to:

drink extra fluids to replace lost blood volume
avoid strenuous exercise for the remainder of the day of donation. The latter
advice is given to prevent fainting and to minimize the possibility of hematoma
development at the venipuncture site.
Other complications:
Thrombophlebitis at site of injection
Pain and bruising = = =
Air-embolism: rare, in bottle packs(historical)
Storage changes of blood components:
a progressive loss of viability of the red cells, and depletion of (2,3 - DPG) & ATP.
potassium gradually leaks out through the cell membrane sodium seeps in.
The pH of blood decreases with storage,
The osmotic fragility of the red blood cells gradually increases
number of changes in platelet function that occur during platelet storage, including:
increased platelet activation
decreased responsiveness to agonists such as ADP
spontaneous lysis, of RBCs  rise of plasma hemoglobin levels.
Because some residual leukocytes are invariably present, various cytokines are also
found in stored blood, and these may play a role in some transfusion reactions.

Acceptable limits for the recovery of red cells and platelets following transfusion are as
follows:
> 75% of red cells/ 24 hours following transfusion,
> 66% of platelets (compared with that of fresh platelets).
 ADVERSE BLOOD
REACTIONS(TRANSFUSION
REACTIONS( REACTIONS)


any unfavorable transfusion-related
event occurring in a patient during or
after transfusion of blood components
 Blood Transfusion: Immediate Reactions
1. Acute Haemolytic Transfusion Reactions
2. Febrile Non-Haemolytic Transfusion Reactions
3. Allergic Reactions:
1. Anaphylaxis
2. Skin Reaction
4. Transfusion-related Acute Lung Injury
5. Bacterial Contamination
6. Circulatory Overload
7. Physically or Chemically Induced Transfusion
Reactions (PCITR’s)
Blood Transfusion: Delayed Reactions
1. Delayed Haemolytic Transfusion Reactions
2. Post- transfusion Purpura
3. Infection Transmission
4. Transfusion-related Graft-versus-Host Disease
5. Immune Modulation
6. Iron Overload
IMMEDIATE BLOOD TRANSFUSION REACTIONS:

ACUTE HAEMOLYTIC
TRANSFUSION

REACTIONS
Intra-vascular
 Extra-vascular
Immediate Blood Transfusion Reactions:
Acute Intra-vascular Haemolytic Transfusion Reactions
Trigger: ABO antigens on transfused red cells Not
shared by the Recipient
Reactor: Anti-A or Anti-B of Ig M type
Pathophysiology
Full Complement Cascade Activation
1. Complement Components C3a,C5a
2.Cytokines: IL-1, IL-6,IL-8, TNF
3.Free Haemoglobin.
4.DIC
The underlying cause of IHTR is transfusion of an incompatible whole
blood or RBC product to a recipient.
The four most commonly identified RBC antibodies causing IHTR are:
anti-A,
anti-Kell,
anti-Jka,
anti-Fya.
IMMEDIATE BLOOD TRANSFUSION REACTIONS:
ACUTE INTRA-VASCULAR HAEMOLYTIC TRANSFUSION
REACTIONS
Clinical Picture
 Fever, Flushing, Rigors
 Headache
 Heat or pain at cannulated vein
 Restlessness Diagnosis
 Bronchospasm Clinical picture
Red urine
 Hypotension Red plasma
 Back or loin pain Lab Confirmation
 Oozing in the surgical field
 Red urine ( haemoglobinuria )
 Oliguria or anuria
IMMEDIATE BLOOD TRANSFUSION REACTIONS:
ACUTE INTRA-VASCULAR HAEMOLYTIC TRANSFUSION
REACTIONS
Laboratory Workup
 Obtain Blood and urine samples, inspect color
 Check paper work
 Repeat cross Match
 CBC
 Direct Coombs’ test
 DIC screen: PT,PTT, Fibrinogen
 BUN, Cr, electrolytes
 Haemolysis screen: LDH, Haptoglobin
 Blood culture if sepsis is suspected
IMMEDIATE BLOOD TRANSFUSION REACTIONS:
ACUTE INTRA-VASCULAR HAEMOLYTIC TRANSFUSION
REACTIONS
Management
 Stop transfusion Immediately
 Replace giving set, keep IV line with Normal saline
 Check patient ID against donor unit
 Cardio-pulmonary support
 Insert urine cath. And start Forced Diuresis
( ensure 100 ml/h for 24 h to get rid of free Hb)

Outcome
Mortality ~ 10 %
 IMMEDIATE BLOOD TRANSFUSION REACTIONS:
ACUTE EXTRA-VASCULAR HAEMOLYTIC
TRANSFUSION REACTIONS
Trigger: Rh antigens not shared by the
patient
Reactor: Anti-Rh antibodies of Ig G
type
Response: Pathophysiology
 Incomplete complement
activation
 Coating of transfused red cells
with C3b
 Extravascular phagocytosis by
RES
 Cytokines from activated RES
 IMMEDIATE BLOOD TRANSFUSION REACTIONS:
ACUTE EXTRA-VASCULAR HAEMOLYTIC
TRANSFUSION REACTIONS
Clinical Features
 Less severe, may be no signs
 Onset > I hour
 Fever
 + Jaundice
 Rarely Haemoglobinuria or renal dysfunction

Laboratory: Anti-complementary Coombs positive

Managment
Stop Transfusion
Supportive
Mortality very rare
IMMEDIATE BLOOD TRANSFUSION REACTIONS:
FEBRILE NON-HAEMOLYTIC TRANSFUSION REACTION
( FNHTR)
Trigger : Leucocyte antigens on infused blood not shared by the patient
Reactors: Leuco-agglutinins in the patient from previous exposure

Pathophysiology
Cytokine released from the transfused activated leucocytes

Clinical Features

Fever after 30-90 min

+ Rigors
+ Headache
No Hypotension
No Bronchospasm
No flank pain
No haemoglobinaemia
No Haemoglobinuria
IMMEDIATE BLOOD TRANSFUSION REACTIONS:
FEBRILE NON-HAEMOLYTIC
TRANSFUSION REACTION ( FNHTR)
Management
 If Temp < 40 + Stable patient:
 Stop transfusion
 Antipyretics ( No rule of Anti-histamines )
 Check the bag and cross match
 Exclude red urine or red plasma
 Resume transfusion at a slower rate
 If recurrent: Leucodepleted transfusion in the future

If Temp 40 or more + Unstable patient:

Stop transfusion
Manage as possible acute haemolytic reaction till lab.
Confirmation or exclusion.
Immediate Blood Transfusion Reactions:
Febrile Non-Haemolytic Transfusion Reaction ( FNHTR)

Prevention/ Recurrence of FNHTR’s:

 pre-transfusion administration of antidotes

 Antipyretics such as aspirin or acetaminophen can be used to

premedicate a patient before transfusion.
 use leukocyte-depleted blood

 removal of buffy coat

 sedimentation

 red cell washing

 use of micro-aggregate filtration (leukoreduction)

IMMEDIATE BLOOD TRANSFUSION REACTIONS:
TRANSFUSION- RELATED ACUTE LUNG INJURY ( TRALI)
Sudden onset of acute respiratory distress within 6 hours( u.
1-2h) of transfusion
Rare: 1/5000 transfusions
Pathophysiology
Trigger: Leucoagglutinins in the bag against patient’s leucocytes
Reactors: Patient leucocytes
Result: massive Leucocyte activation
 Cytokine storm
 Pulmonary Endothelial and Epithelial Injury  ARDS

Management
Clinical Features
Fever, chills Cardio-Pulmonary Support
Acute Respiratory Distress Steroids
Normal CVP (Central Venous Pressure) Diuretics of No value
CXR: Pulmonary Infiltrate Mortality
High
PATHOPHYSIOLOGY OF
(TRALI)
Leukocyte Ab in donor react with pt. leukocytes

Activate complements

Adherence of granulocytes to pulmonary endothelium with

release of proteolytic enz.& toxic O2 metabolites

Endothelial damage

Interstitial edema and fluid in alveoli

Immediate Blood Transfusion Reactions:
Allergic Acute Transfusion Reactions
Pathophysiology
 Trigger: Plasma proteins in the transfused blood
 Reactors: Patient antibodies of IgE type
 Response:
 Mast cell degranulation
 + Complement Activation
 + Cytokines
IMMEDIATE BLOOD TRANSFUSION REACTIONS:
ALLERGIC ACUTE TRANSFUSION
REACTIONS
Clinical Features
 Mild / Skin-restricted ( common: 1%):
 Pruritus, Uerticaria, No fever or Hypotension
 Severe / Systemic ( Anaphylaxis):
 As above +
 Fever
 Hypotension
 Bronchospasm, Angio-edema
 Immediate Blood Transfusion Reactions:
Allergic Acute Transfusion Reactions
Management
 Mild / Skin-restricted :
 Stop transfusion temporary
 Anti-histamines
 Resume Transfusion
Severe / Systemic ( Anaphylaxis):
Stop transfusion
Anti-histamines ( H1+H2 blockers)
Epinephrine: 1 ml of 1/1000 IM
Hydrocortisone 100 mg IV
Cardio-pulmonary support
 IMMEDIATE BLOOD TRANSFUSION REACTIONS:
ACUTE PYROGENIC TRANSFUSION
REACTIONS
Pathophysiology
 Trigger: Bacterial Pyrogens/Endotoxins in the transfused
blood contaminated with cold-growing organisms as:
 Pseudomonas
 Yersinia
 Some Staph
 Reactors: Patient Mono-nuclear cells
 Response:
 Cytokine Storm

Management
As Acute Haemolytic reaction
BUT Add Broad- spectrum
Antibiotics
Clinical Features
Like :
Acute Haemolytic reaction BUT:
No Hemoglobinuria
No Hemoglobinaemia
FNHTR BUT More Severe
 Immediate Blood Transfusion Reactions:
Acute Circulatory Overload
 Acute cardiogenic pulmonary edema
 In rapidly transfused, non-bleeding (euovolemic) patients
 More in infants, elderly or cardiac patients

D.D. from other Acute transfusion reactions:

No Fever ( DD from TRALI, FNHTR)
No red urine or plasma and Negative Coombs
( DD from Acute haemolytic reaction)
Prevention
Never exceed 2-3 ml/kg/hour Unless Bleeding
Pre-medicate with Diuretics in Cardiac or severely anemic patients
Management
Diuretics
Consider Haemodialysis
Supportive
IMMEDIATE BLOOD TRANSFUSION REACTIONS:
PHYSICALLY OR CHEMICALLY INDUCED
TRANSFUSION REACTIONS (PCITR’S)

 heterogenous group of conditions including:

 Physical RBC damage
 Depletion and dilution of coagulation factors
and platelets
 Hypothermia
 Citrate toxicity
 Hypokalemia / hyperkalemia
IMMEDIATE BLOOD TRANSFUSION REACTIONS:
PHYSICALLY OR CHEMICALLY INDUCED
TRANSFUSION REACTIONS (PCITR’S)
 Physical Damage to RBC’s
 intravascular lysis due to hypertonic or hypotonic solutions
 heat damage from blood warmers, during shipping, in hot rooms
 freeze damage in absence of cryoprotective agent during
shipping
 Mechanical Damage
 blood pumps, roller pumps
 infusion under pressure through small bore needles
Immediate Blood Transfusion Reactions:
Physically or Chemically Induced Transfusion Reactions
(PCITR’s)


Citrate toxicity

ACD/ CPD has 1.4-1.6 g of citrate - no toxicity
 citrate > 100 mg/ dl - citrate toxicity
 Causes
 ADULTS
 rate of BT > 1 liter/ 10 min or BT volume exceeds 6 L
administered in < 2 hours
 CHILDREN
 exchange transfusion  hypocalcemia
 Immediate Blood Transfusion Reactions:
Physically or Chemically Induced Transfusion Reactions
(PCITR’s)
 Potassium toxicity:
 Mechanism: high potassium load with prolonged blood storage
- hyperkalemia
 Clinical manifestations: cardiac excitability
 ECG findings: peak T waves
 Laboratory findings: hyperkalemia
 Management: calcium gluconate
DELAYED BLOOD TRANSFUSION
REACTIONS
1. Delayed Haemolytic transfusion reactions
2. Post-transfusion Purpura
3. Infection transmission
4. Transfusion GVHD
5. Iron Overload
6. Immune Modulation
 Delayed Hemolytic TransfusionReaction (DHTR)

Definition
DHTR is most often the result of an anamnestic response in a
patient who has previously been sensitized by transfusion,
pregnancy, or transplant and in whom antibody is not detectable
by standard pretransfusion methods.

Clinical
signs and symptoms are usually mild;
severe DHTR cases and fatalities are uncommon.
Unexpected or unexplained decreases in hemoglobin or hematocrit values
following transfusion should be investigated as a possible DHTR.

Pathophysiology
Two different types of DHTR have been identified:
1. Secondary (anamnestic) response to transfused RBCs
2. Primary alloimmunization
In DHTR caused by a secondary response,
about 3 to 7 days from time of transfusion are necessary for enough
antibody to be produced by the patient to cause clinical signs and
symptoms of extravascular RBC hemolysis.
In DHTR caused by primary alloimmunization,
the patient has no past history of pregnancy, transfusion, or transplant

Management
The goal of therapy is to
prevent
Renal function can be
supported with intravenous
fluid therapy to maintain a
normovolemicstatus.
Only symptomatic anemia
should be treated with RBC
transfusions
 Delayed Blood Transfusion Reactions
Post-Transfusion Purpura (PTP)
 Consists of profound thrombocytopenia occurring 1-2 weeks after
transfusion

 Pathophysiology:
Effect of antibody directed against donor platelet
antigens that the recipient lacks
Commonly associated with human platelet-
specific alloantigen 1a (HPA-1a)
 Delayed Blood Transfusion Reactions
Post-Transfusion Purpura (PTP)
 Treatment and Prevention of PTP:
IVIG
plasmapheresis
steroids
avoidance of antigen-positive platelet
transfusion with previous PTP
DELAYED BLOOD TRANSFUSION REACTION
TRANSFUSION-ASSOCIATED GRAFT-VERSUS-HOST
DISEASE ( TA-GVHD)

Pathophysiology
Infusion of Immunocompetent Cells
(Lymphocyte)

Patient at risk

proliferation of donor T lymphocytes

attack against patient tissue

Delayed Blood Transfusion Reaction
Transfusion Associated Graft versus Host Disease (TA-GVHD)

 Clinical Manifestations of GVHD:

 onset: 7-10 days from transfusion
 fever
 reddish, raised rash spreading from trunk or face to extremities
- bullous lesions  erythroderma
 hepatitis
 watery diarrhea
 non-specific signs: anorexia, nausea and vomiting
Delayed Blood Transfusion Reaction
Iron Overload
 common in patients with chronic diseases requiring multiple and
prolonged transfusions (thalassemia)
 on the average, 1 unit PRBC = 200 mg iron

 also “transfusion hemosiderosis”

 chronic iron overload leads to hepatic, cardiac and pancreatic

disease.
 prevention:

iron chelation therapy (desferoxamine)

Delayed Blood Transfusion Reaction
Alloimmunization
 Pathophysiology:
 after first exposure to donor antigen  recipient
memory lymphocytes are invoked  moderate
production of IgG and IgM
 on second exposure to donor antigen  rapid and large
production of IgG within the first 2 days

Clinical manifestations:
mild to severe
Laboratory tests:
antibody screening
Treatment of Alloimmunization:
Accurate matching of donor and recipient RBC phenotypes
MASSIVE BLOOD
TRANSFUSION
Definition
Transfusion of Blood ~ Blood Volume within 24 hours
•20 units whole blood
•10 units packed cells
Complications Complications
• Dilutional • Dilutional Thrombocytopenia
Thrombocytopenia
•Common after 10 units
•Dilutional Coagulopathy
•Severe after 20 units
•Metabolic
•Give platelet transfusion if <
•Hypothermia 80,000 + bleeding
MASSIVE BLOOD
TRANSFUSION
Complications
•Dilutional Coagulopathy
•Particularily if blood stored > 2 weeks
•Monitor Coagulation profile
•FFP if Abnormal lab
•DIC is Rare
•Metabolic: Citrate Intoxication
•Acidosis, Hypocalacemia, Hyperkalaemia
•Rare Except in Infants or Hepatic patients
 Haemovigilance
derived from the Greek word ‘hema’ = blood and the Latin word ‘vigilans’ =
watchful.
defined as a set of surveillance procedures covering whole transfusion chain from
the collection of blood and its components to the follow up of its recipients,
intended to collect and access information on unexpected or undesirable effects
resulting from the therapeutic use of labile blood products, and to prevent their
occurrence and recurrence.Thus, the ultimate goal of a hemovigilance system
is to improve the safety of blood transfusion.

 started in France in 1994 

The UK’s professional haemovigilance system: SHOT,(Serious Hazards Of
Transfusion) was introduced in 1996 with voluntary reporting of adverse
transfusion reactions, errors and also near-miss events.
In Canada: Transfusion Transmitted Injuries Surveillance System [TTISS]),
Netherlands (Transfusion Reactions in Patients [TRIP]),
At present, on global scale an International Hemovigilance Network (IHN) is
functioning which evolved from European Hemovigilance Network (EHN—founded
in 1998).
Scope of Hemovigilance
A. reporting of:
1.adverse reaction versus adverse events,
2.all versus serious adverse reactions only;
3.only incidents in recipients or also in donors;
4.all adverse events or only the serious adverse reactions in recipients.

B. cover processes throughout the entire transfusion chain, from blood donation,
processing, and transfusion to patients for the monitoring, reporting, and
investigation of adverse events and reactions and near misses related to blood
transfusion.

It should be well coordinated between the blood transfusion service, hospital

clinical staff and transfusion laboratories, hospital transfusion committees,
regulatory agency, and national health authorities

Can be:
Recipient hemovigilance
Donor =
Appropriate use of blood and alternativesvto allogeneic
blood transfusion

reasons for aiming to reduce unnecessary allogeneic

blood transfusions:
1 safety of the patient, by avoiding errors, as well as
microbiological and immunological risks;
2 increasing difficulties in the recruitment of blood
donors;
3 cost containment, with the ever-increasing demands by
the regulatory agencies and technological advances;
4 high anxiety levels in patients, disproportionate to the
real residual infectious risk of transfusion.
Patient blood management (PBM)
 Defined as an evidence-based multidisciplinary approach
to optimizing the care of patients who might need
transfusion, considering: alternatives, where feasible, and
giving transfusion only when appropriate.

 Covers all aspects of decision-making in transfusion

therapy, including initial patient evaluation with use of
appropriate indications and transfusion triggers, minimizing
blood loss and optimization of the patient’s haemoglobin
wherever possible.

 The application of the principles of PBM can reduce the

need for transfusion and therefore health-care costs, while
ensuring that limited donor blood supplies are available for
the patients who need them most.
Clinical decision to transfuse
Depends on:
1.Laboratory parameters ( Hb, platelet count or clotting screen,
2.Careful assessment of the patient’s overall clinical status.
 STEM CELL
TRANSPLANTATION
(SCT)
Principles
Eliminating a patient’s haemopoietic and
immune system by chemotherapy and/ or
radiotherapy and replacing it with stem
cells either from another individual or with
a previously harvested portion of the
patient’s own haemopoietic stem cells.

Depending on the source of the stem

cells the term includes:
1.Bone marrow transplantation (BMT),
2.Peripheral blood stem cell (PBSC)
transplantation
3.Umbilical cord transplantation.

Depending on the type of donor, SCT

may be
1.Syngeneic,
2.Allogeneic.
3. Autologous.
Collection of stem cells
1. Peripheral blood stem cell
collection
This is now the preferred source of stem cells
for both autologous and allogeneic
transplantation.
Peripheral blood stem cells (PBSCs) are
taken using a cell-separator machine
connected to the patient or donor via
peripheral cannulae
normally contains too few haemopoietic stem
cells to allow collection of sufficient numbers
for transplantation.
Growth factors can increase the number by
around 10–100 times. PBSC collections are
then taken and, depending on the efficiency of
stem cell mobilization, repeated collections
may be needed for up to 3 days.
The adequacy of the collection is assessed by
CD34+ cell count.
Generally > 2.0 x 10*6/kg CD 34+ cells are
needed for transplantation.
2. Bone marrow collection
The donor is given a general anaesthetic
and 500–1200 mL of marrow is harvested
from the pelvis. The marrow is
anticoagulated and a mononuclear cell
count is taken to assess the yield, which
should be approximately 2–4 x 10*8
nucleated cells/kg body weight of the
recipient.
3. Umbilical cord blood
Fetal blood is a rich source of haemopoietic
stem cells which may be collected from cord
blood. Because of the relatively small
numbers of stem cells collected from a single
cord.
Most useful for children who do not have a
fully matching sibling or unrelated donor.
Less stringent HLA-matching is needed.
Double cord donations may be needed to
obtain sufficient stem cells for adult
recipients.
Immune reconstitution is slower after cord
blood transplantation.
Stem cell processing

After collection, the stem cell harvest can be

processed with removal of red cells and
concentration of the mononuclear cells.
In some protocols antibodies to remove T
lymphocytes are used to reduce the risk of
graft-versus-host disease (GVHD).
Autologous collections may be ‘purged’ by
chemotherapy or antibodies in an attempt to
remove residual malignant cells.
CD34+ stem cells may be selected from both
types of harvest.

STEPS OF SCT
1.Donor selection
2.Stem cell harvest/Processing
3.Conditioning of patient
4.Stem cell transplantation
5.Post-transplant supportive care and
check for engraftment.
STEPS OF SCT

1.Donor selection
2.Stem cell
harvest/Processing
3.Conditioning of patient
4.Stem cell transplantation
5.Post-transplant supportive
care and check for
engraftment.
Conditioning
designed to: Stem cell
1. Eradicate the patient’s haemopoietic and
immune system transplantation:
2. Eradicate, if present, malignancy.
3. suppressing the host immune system, it On day (Zero)
helps to prevent rejection of the ‘foreign’ stem Simply by IV infusion through a
cells. central venous cath.
Takes not > hr
Can be:
1.Myeloablative conditioning regimens
irreversibly destroy the haemopoietic function
of the bone marrow with high doses of
chemotherapy or radiotherapy. At least 36
hours are allowed for the elimination of the
drugs from the circulation following the last
dose of chemotherapy before donor stem cells
are infused.
2. Non-myeloablative conditioning regimens
to reduce the morbidity and mortality of
allogeneic transplantation. These do not
completely destroy the host bone marrow.
Post-transplant engraftment and immunity

After a period of typically 1–3 weeks of severe pancytopenia, the first signs of
successful engraftment are:
1. Appearance of monocytes and neutrophils in the blood with a subsequent
increase in platelet count.
2. Reticulocytosis also begins.

G-CSF may be used to reduce the period of neutropenia.
 Engraftment is usually quicker following PBSC transplantation compared with
BMT.

The marrow cellularity gradually returns to normal but the marrow reserve
remains impaired for 1–2 years.

There is profound immunodeficiency for 3–12 months with a low level of CD4
helper cells.
Immune recovery is quicker after autologous and syngeneic SCT than following
allogeneic SCT.

The patient’s blood group changes to that of the donor and antigen-specific
immunity becomes that of the donor after approximately 60 days.
Apheresis(hemapheresis)
(Greek= “to separate or remove.”)
blood is withdrawn from a donor or patient and separated into
its components.
One (or more) of the components is retained, and the
remaining constituents are recombined and returned to the
individual.
Any of the components of blood can be removed, and the
procedures are specified by the component selected.
Plasmapheresis.
Plateletpheresis (thrombocytapheresis)
Red blood cells (erythrocytapheresis)
Leukocytes (leukapheresis)
PRINCIPLES OF APHERESIS:

1. The centrifugation method:

most commonly used instruments employ it:
For cytapheresis + plasmapheresis, both therapeutically &
collectively.
can be divided into two basic categories
a. Intermittent flow centrifugation (IFC)
b. Continuous flow centrifugation (CFC)

2. The filtration techniques:

Occasionally used
Only for therapeutic plasmapheresis
Intermittent flow centrifugation :
Performed in cycles (also called passes) .Blood is drawn from
an individual with the assistance of a pump. To keep the blood
from clotting, an anticoagulant is added to the tubing.
The blood is pumped into a centrifuge bowl through the inlet
port. The bowl rotates at a fixed speed, separating the
components according to their specific gravities. A rotary seal is
used, resulting in a closed system.
The RBCs, which have greater mass, are packed against the
outer rim of the bowl, followed by the WBCs, platelets, and
plasma.
The separated component(s) flow from the bowl through the
outlet port and are harvested as desired into separate collection
bags .The undesired components are diverted into a reinfusion
bag and returned to the individual. Reinfusion completes one
cycle. The cycles are repeated until the desired quantity of
product is obtained (e.g., a plateletpheresis procedure usually
takes 6 to 8 cycles to collect a therapeutic dose.
One of the advantages of the IFC procedure is that it can be
done with only one venipuncture (one-arm procedure; that is,
the blood is drawn and reinfused through the same needle).
Continuous flow centrifugation
withdraw, process, and return the blood to
the individual
simultaneously(uninterrupted, or
continuous).
Because blood is drawn and returned
continuously during a procedure, two
venipuncture sites are necessary.
Blood is drawn from the phlebotomy site
with the assistance of a pump, mixed with
anticoagulant, and collected in a chamber
or belt, depending on the machine.
 Separation of the components is achieved
through centrifugation.
 Comparison between IFC and CFC machines

CFC IFC Item

Larger Smaller/more portable Machine

Two Single Venepuncture site

Less Greater Extracorporeal volume

Lower Higher Risk of hypovolemia

Less More Time of operation

Membrane filtration technology
 Can also be used to separate blood
components.
 Blood that passes over membranes with
specific pore sizes allows passage of
plasma through the membrane while the
cellular portion passes over it.
 Has several advantages over
centrifugation, including:
1. The collection of a cell-free product
2. The ability to selectively remove
plasma components by varying the pore
size
Therapeutic Apheresis
An important modality of therapy in the management of several diseases
Plasmapheresis: the selective removal of plasma. This includes the
collection of plasma from normal donors.
Therapeutic plasma exchange: the removal of a large proportion of a
patient’s plasma and replacement with crystalloid or colloid fluids
and/or plasma product
Therapeutic cytapheresis, the removal of red cells, leukocytes or
platelets: used infrequently in the management of patients with
hemoglobinopathies, leukostasis, or thrombocytosis, respectively.
Evidence-based guidelines for therapeutic apheresis

In 2013, the American Society for Apheresis published the 5th Special

Edition of evidence based guidelines for the practice of Apheresis Medicine.
These guidelines are based upon a systematic review of available scientific
literature. Clinical utility for a given disease is denoted by assignment of
an ASFA Category (I – IV). The quality and strength of evidence are denoted
by standard GRADE recommendations.

ASFA Categories are defined as follows:

Category I for disorders where therapeutic apheresis is accepted as a first line

treatment,
Category II for disorders where therapeutic apheresis is accepted as a second-
line treatment,
Category III for disorders where the optimal role of therapeutic apheresis is not
clearly established and
Category IV for disorders where therapeutic apheresis is considered ineffective
or harmful.
TPE for immunologic diseases: 1.0 to 1.5 plasma volumes exchanged at each
procedure; repeated, for a total of 5 to 7 exchanges over a period of 7 to 10
days.
After exchange of one plasma volume, approximately 62% of the original
plasma has been removed. The efficiency of plasma exchange decreases with
further exchange.
Efficacy varies with the plasma factor to be removed:
IgM and fibrinogen: largely confined to IV space:, removed most efficiently .
IgG is removed less efficiently: because only 45 to 50% of the body’s IgG is
located in IV space.
Therapeutic leukapheresis
Used in the treatment of patients with
leukostasis/Hyperleucocytosis:
An emergency measure in: Esp. in pregnancy
Acute leukemia
CML To prevent
Pul & cerebral leukostasis and stroke
CLL Tumor lysis syndrome
Impaired capillary flow
Sezary syndrome

Collection of leukocytes by apheresis, followed by ex vivo exposure of the

leukocytes to UV light in the presence of a psoralen, and reinfusion of the treated
leukocytes (“photopheresis”) has become standard therapy for cutaneous T-cell
lymphoma. This procedure may be useful in the treatment of other disorders such
as GVHD .
Therapeutic plateletpheresis
performed very rarely
not considered to be first-line therapy.
indicated in patients with very high platelet counts, usually >1.0 to 1.5 ×
1012/L, with the possibility of serious thrombotic or hemorrhagic
problems.
Must be followed by cytotoxic therapy to prevent rebound after the
procedure, or its effect is very short-lived.
Erythrocytapheresis (red cell exchange):
used in sickle cell disease to replace sickled cells with normal
erythrocytes and thereby prevent thromboses and improve capillary
circulation .
Stroke and acute chest syndrome are the two major indications for red
cell exchange in patients with sickle disease.
Therapeutic Absorption of Plasma Constituents :
Selectively remove constituents of plasma implicated in disease
processes.
patient’s plasma is withdrawn and separated by apheresis
technology, passed over a selective absorption column, and
reinfused into the patient.
used to selectively remove IgG (staphylococcal protein A
columns) or low-density-lipoprotein (LDL) cholesterol in
familial hypercholesterolemia.
Adverse Effects
common but usually mild. Include:
1.vasovagal reactions,

2. fluid imbalance with hypovolemia or overload,

3. fever, chills,

4. hypocalcemic citrate reactions ranging from paresthesias to

arrhythmias.
5.If plasma is used as the replacement fluid, urticarial reactions may
be encountered.
6.Mild dilutional coagulopathy and thrombocytopenia occur but are
rarely significant .
7. Problems related to venous access are common.

8. The possibility of immunologic rebound after TPE

9. Infections related to indwelling venous lines are not uncommon.

10. Deaths have been reported with therapeutic apheresis .

Viral blood transmitted disease

A number of diseases have the potential to be

transmitted by transfusion of blood or its
components. The viruses that pose the greatest
potential risk for transmission by transfusion
 1. Hepatotropic virus
HAV ( rarely transmitted by transfusion )
HBV (readily transmissible by blood )
HCV (readily transmissible by blood )
2. Retroviruses
HIV1 and HIV2 (readily transmissible by
VIRUSES blood)
:

3. HERPESVIRUSES: CMV and EPV

4.Others like parvovirus B19 and West

Nile virus
 Hepatitis viruses :
Donors with a history
of hepatitis are
deferred for 12 months.
The hepatitis viruses
can be transmitted in
cellular and plasma
components, as well as
plasma products.
HBV:
All donations are tested for the presence of HBsAg by
sensitive enzyme - linked immunosorbent assays
(ELISAs) that can detect at least 0.2 IU/mL of HBsAg.
HBsAg - positive subjects are permanently excluded
from donation, and should be under specialist follow -
up, As they have an increased risk of developing chronic
liver disease and hepatocellular carcinoma.
HBsAg testing will not exclude all donors capable of
transmitting HBV.
The presence of IgM
anti-HAV antibody is
required for diagnosis of
hepatitis A. IgM
antibodies are
detectable at or prior to
the onset of clinical
illness and decline in 3
to 6 months.
IgG antibodies to HAV
appear soon after IgM
and may persist for
years after the infection.

IgM anti-HBv is the first antibody to appear, and it persists for about 6 months.
Appearance of this antibody indicates current or recent acute infection.
IgG anti-HBv appears after IgM and these together used for initial diagnosis and
follow up the course of disease.
 HCV
Readialy transmissible by blood Incubation period range
from 2-28 weeks.
The acute phase infection is often without symptoms or
mild. Diagnosis by detection of antibodies or virus RNA
(ribonucleic acid) and/or antigen in serum.

Individuals with a history of jaundice may be

accepted as donors, provided that they have been
shown to be negative for markers of HBV and HCV.
Clinical jaundice may be due to causes other than
HBV and HCV, including non - viral causes.
 HAV rarely transmitted by blood transfusion (usually
transmitted by feco-oral rout),it is usually transmitted by
HAV clotting factor concentrates.
I.P 28 days on average and peak viraemic period occurs 2
WK before the onset of elevation of liver enzyme or the
appearance of jaundice.
HAV cause mild to sever symptoms and hepatitis A vaccine is
effective for prevention.
 can be transmitted in both cellular and plasma
HIV1H components.
IV2 Screening tests : nucleic acid testing for HIV
RNA (NAT) , Ag/Ab serological testing

HTCLV1 usually associated with 2 distinct clinical

condition:
1. Adult T-cell leukaemia (I/P 20Yrs)
2. Tropical spastic paraparesis (rare)
HTCLV1 HTCLV2 its importance is not clear.
HTCLV2 Both HTCLV1, HTCLV2 are cell associated and not
transmitted in plasma.
Routine serological screening is by ELISA, but
confirmation of positive results can still cause
difficulties because other retroviruses may cross -
react
WIDOW
PERIOD

Is the time between first infection and

when the test can reliably detect the
infection in antibody depend testing,
the window period is dependent on
the time taken for seroconversion.

the window period has been

estimated to be less than 2 weeks on
average. With the recently introduced
nucleic acid test (NAT) for HIV
RNA, the window period has
decreased to approximately 7 days.
  Most cases of post - transfusion cytomegalovirus(CMV)
infection are subclinical, the syndrome of post –
transfusion infectious mononucleosis - like illness is well
recognized, especially after the transfusion of large
amounts of blood , CMV is cell associated .
The infection is characterized by fever, splenomegaly
and atypical lymphoid cells in the peripheral blood, with a
negative Paul Bunnell test.
CMV

No necessity to screen all donors for evidence of past infection .

There are group of particular risk such as
1- Premature babies.
2-Pregnant women
3- Bone marrow and other organ transplant
recipients.
Lack of evidence of past CMV infection in these individuals anti
CMV free blood and blood components should be provided.
Haemolytic disease of the newborn
The result of red cell alloimmunization in which IgG antibodies passage from the
maternal circulation across the placenta into the circulation of the fetus where they
react with fetal red cells and lead to their destruction.

Anti‐D antibody is responsible for most cases of severe HDN

anti‐c,
anti‐E,
found in occasional cases
anti‐K
a wide range of other antibodies
Although antibodies against the ABO blood group system are the most frequent
cause of HDN this is usually mild.
Rh haemolytic disease of the
newborn
When an Rh D‐negative woman has
a pregnancy with an Rh D‐positive
fetus, Rh D‐positive fetal red cells
cross into the maternal circulation
(especially at parturition and during
the third trimester) and sensitize the
mother to form anti‐D.
Anti‐D crosses the placenta to the
fetus during the next pregnancy,
coats RhD‐positive fetal red cells
and results in reticuloendothelial
destruction of these cells, causing
anaemia and jaundice. If the father is
heterozygous for D antigen, and
mother RhD negative there is a 50%
probability that the fetus will be D‐
positive. The fetal Rh D genotype
can be established by polymerase
chain reaction (PCR) analysis for the
presence of Rh D in a maternal
blood sample.
Prevention of Rh immunization

At the time of booking:

ABO and Rh group

antibodies screen For all pregnant women
at least twice during the pregnancy.

All non‐sensitized Rh D‐negative women: 500 units (100 μg) of anti‐D IM at 28

and 34 weeks’ gestation to reduce the sensitization from fetomaternal
haemorrhage.

Fetal Rh D typing from DNA in maternal blood can be used before 28 weeks.
If the fetus is Rh D‐negative, no further anti‐D prophylaxis is needed.

At birth the babies of Rh D‐negative women who do not have antibodies must have
their cord blood grouped for ABO and Rh. If the baby’s blood is Rh D‐negative, the
mother will require no further treatment.
If the baby is Rh D‐positive, prophylactic anti‐D: 500 units IM within 72 hours of
delivery.
Kleihauer test is performed. This
uses differential staining to estimate the
number of fetal cells in the maternal
circulation.

If the Kleihauer is positive many centres

will perform flow cytometry for a more
accurate estimate of the volume of feto‐
maternal haemorrhage (FMH).

The chance of developing antibodies is

related to the number of fetal cells found.
The dose of anti‐D is increased if there is
greater than 4 mL transplacental
haemorrhage. Anti‐D IgG (125 units) is
given for each 1 mL of FMH greater than 4
mL.
Potentially Sensitizing
episodes during
pregnancy

Anti‐D IgG should be given to

Rh D‐negative women who
have potentially sensitizing
episodes during pregnancy:

250 units is given if the event

occurs up to week 20 of
gestation and 500 units
thereafter, followed by a
Kleihauer test.
Clinical features of HDN
1.Severe disease
2. Intrauterine death from
hydrops fetalis

2. Moderate disease
The baby is born with anaemia and jaundice and may show pallor, tachycardia,
oedema and hepatosplenomegaly. If the unconjugated bilirubin is not controlled
and reaches levels exceeding 250 μmol/L, bile pigment deposition in the basal
ganglia may lead to kernicterus– central nervous system damage with generalized
spasticity and possible subsequent mental deficiency, deafness and epilepsy. This
problem becomes acute after birth as maternal clearance of fetal bilirubin ceases
and conjugation of bilirubin by the neonatal liver has not yet reached full activity.

3. Mild disease
Mild anaemia with or without jaundice.
Investigations :variable anaemia
high reticulocyte count;
the baby is Rh D‐positive,
DAT is positive
serum bilirubin raised.
In moderate and severe cases:
many erythroblasts are seen in the blood film
(erythroblastosis fetalis.)
Treatment of established
anti‐D sensitization
If anti‐D antibodies are detected during
pregnancy they should be quantified at
regular intervals.

The clinical severity is related to:

The strength of anti‐D present in
maternal serum
The IgG subclass,
The rate of rise of antibody
 Past history.
The development of haemolytic disease in
the fetus can be assessed by velocimetry
of the fetal middle cerebral artery by
Doppler ultrasonography, as increased
velocities correlate with fetal anaemia.

If anaemia is detected, fetal blood sampling and intrauterine transfusion of

irradiated Rh D‐negative packed red cells may be indicated
Treatment
Exchange transfusion may be
necessary; the indications
severe anaemia (Hb <100 g/L at
birth)
severe or rapidly rising
hyperbilirubinaemia.
More than one exchange transfusion
may be required and 500 mL is
usually sufficient for each exchange.
The donor blood should be:

Phototherapy (exposure of the infant

to bright light of appropriate
wavelength) degrades bilirubin and
reduces the likelihood of kernicterus.
ABO haemolytic disease of the newborn
In 20% of births, a mother is ABO incompatible with the fetus. Group A and group
B mothers usually have only IgM ABO antibodies .
The majority of cases of ABO HDN are caused by ‘immune’ IgG antibodies in
group O mothers.
Although 15% of pregnancies in white people involve a group O mother with a
group A or group B fetus, most mothers do not produce IgG anti‐A or anti‐B and
very few babies have severe enough haemolytic disease to require treatment.
Exchange transfusions are needed in only 1 in 3000 infants. The mild course of
ABO HDN is partly explained by:
1.the A and B antigens not being fully developed at birth
2.partial neutralization of maternal IgG antibodies by A and B antigens on other
cells, in the plasma and tissue fluids.

In contrast to Rh HDN, ABO disease

may be found in the first pregnancy and may or may not affect subsequent
pregnancies.
DAT on the infant’s cells may be negative or only weakly positive.
Examination of the blood film shows autoagglutination spherocytosis,
polychromasia and erythroblastosis.